High density molecular arrays on porous surfaces

ABSTRACT

The present invention provides a unique and highly accurate method for generating molecular arrays of very high density on porous surfaces. The method involves the application of focused acoustic energy to each of a plurality of fluid-containing reservoirs to eject a small fluid droplet—on the order of 1 picoliter or less—from each reservoir to a site on a porous substrate surface. High density molecular arrays are provided as well, in which greater than about 62,500 molecular moieties, serving as array elements, are present on a porous surface. Biomolecular arrays that can be generated using focused acoustic ejection include oligonucleotide arrays and peptidic arrays.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 09/727,392, filed Nov. 29, 2000, which is a continuation-in-part ofU.S. patent application Ser. No. 09/669,996, filed Sep. 25, 2000, thedisclosures of which are incorporated by reference herein.

TECHNICAL FIELD

[0002] This invention relates generally to the use of focused acousticenergy in the preparation of molecular arrays, and more particularlyrelates to acoustic ejection of fluid droplets to prepare high densitymolecular arrays.

BACKGROUND

[0003] The discovery of novel materials having useful biological,chemical and/or physical properties often leads to emergence of usefulproducts and technologies. Extensive research in recent years hasfocused on the development and implementation of new methods and systemsfor evaluating potentially useful chemical compounds. In thebiomacromolecule arena, for example, much recent research has beendevoted to potential methods for rapidly and accurately identifying theproperties of various oligomers of specific monomer sequences, includingligand and receptor interactions, by screening high density arrays ofbiopolymers including nucleotidic, peptidic and saccharidic polymers.

[0004] For biological molecules, the complexity and variability ofbiological interactions and the physical interactions that determine,for example, protein conformation or structure other than primarystructure, preclude predictability of biological, material, physicaland/or chemical properties from theoretical considerations at this time.For non-biological materials, including bulk liquids and solids, despitemuch inquiry and vast advances in understanding, a theoretical frameworkpermitting sufficiently accurate prediction de novo of composition,structure and synthetic preparation of novel materials is still lacking.

[0005] Consequently, the discovery of novel useful materials dependslargely on the capacity to make and characterize new compositions ofmatter. Of the elements in the periodic table that can be used to makemulti-elemental compounds, relatively few of the practicallyinexhaustible possible compounds have been made or characterized. Ageneral need in the art consequently exists for a more systematic,efficient and economical method for synthesizing novel materials andscreening them for useful properties. Further, a need exists for aflexible method to make compositions of matter of various material typesand combinations of material types, including molecular materials,crystalline covalent and ionic materials, alloys, and combinationsthereof such as crystalline ionic and alloy mixtures, or crystallineionic and alloy layered materials.

[0006] The immune system is an example of systematic protein and nucleicacid macromolecular combinatorial chemistry that is performed in nature.Both the humoral and cell-mediated immune systems produce moleculeshaving novel functions by generating vast libraries of molecules thatare systematically screened for a desired property. For example, thehumoral immune system is capable of determining which of 10¹²B-lymphocyte clones that make different antibody molecules bind to aspecific epitope or immunogenic locale, in order to find those clonesthat specifically bind various epitopes of an immunogen and stimulatetheir proliferation and maturation into plasma cells that make theantibodies. Because T cells, responsible for cell-mediated immunity,include regulatory classes of cells and killer T cells, and theregulatory T cell classes are also involved in controlling both thehumoral and cellular response, more clones of T cells exist than of Bcells, and must be screened and selected for appropriate immuneresponse. Moreover, the embryological development of both T and B cellsis a systematic and essentially combinatorial DNA splicing process forboth heavy and light chains. See, e.g., Therapeutic Immunology, Eds.Austen et al. (Blackwell Science, Cambridge Mass., 1996).

[0007] Recently, the combinatorial prowess of the immune system has beenharnessed to select for antibodies against small organic molecules suchas haptens; some of these antibodies have been shown to have catalyticactivity akin to enzymatic activity with the small organic molecules assubstrate, termed “catalytic antibodies” (Hsieh et al. (1993) Science260(5106):337-9). The proposed mechanism of catalytic antibodies is adistortion of the molecular conformation of the substrate towards thetransition state for the reaction and additionally involveselectrostatic stabilization. Synthesizing and screening large librariesof molecules has, not unexpectedly, also been employed for drugdiscovery. Proteins are known to form an induced fit for a boundmolecule such as a substrate or ligand (Stryer, Biochemistry, 4th Ed.(1999) W. H. Freeman & Co., New York), with the bound molecule fittinginto the site much like a hand fits into a glove, requiring some basicstructure for the glove that is then shaped into the bound structurewith the help of a substrate or ligand.

[0008] Geysen et al. (1987) J Immun. Meth. 102:259-274 have developed acombinatorial peptide synthesis in parallel on rods or pins involvingfunctionalizing the ends of polymeric rods to potentiate covalentattachment of a first amino acid, and sequentially immersing the ends insolutions of individual amino acids. In addition to the Geysen et al.method, techniques have recently been introduced for synthesizing largearrays of different peptides and other polymers on solid surfaces.Arrays may be readily appreciated as additionally being efficientscreening tools. Miniaturization of arrays saves synthetic reagents andconserves sample, a useful improvement in both biological andnon-biological contexts. See, for example, U.S. Pat. Nos. 5,700,637 and6,054,270 to Southern et al., which describe a method for chemicallysynthesizing a high density array of oligonucleotides of chosenmonomeric unit length within discrete cells or regions of a supportmaterial, wherein the method employs an inkjet printer to depositindividual monomers on the support. So far, however, miniaturized arrayshave been costly to make and contain significant amounts of undesiredproducts at sites where a desired product is made. Thus, even in thebiological arena, where a given sample might be unique and thereforepriceless, use of high density biomacromolecule microarrays has metresistance by the academic community as being too costly, as yetinsufficiently reliable compared to arrays made by lab personnel.

[0009] Arrays of thousands or even millions of different compositions ofthe elements may be formed by such methods. Various solid phasemicroelectronic fabrication derived polymer synthetic techniques havebeen termed “Very Large Scale Immobilized Polymer Synthesis,” or“VLSIPS” technology. Such methods have been successful in screeningpotential peptide and oligonucleotide ligands for determining relativebinding affinity of the ligand for receptors.

[0010] The solid phase parallel, spatially directed synthetic techniquescurrently used to prepare combinatorial biomolecule libraries requirestepwise, or sequential, coupling of monomers. U.S. Pat. No. 5,143,854to Pirrung et al. describes synthesis of polypeptide arrays, and U.S.Pat. No. 5,744,305 to Fodor et al. describes an analogous method ofsynthesizing oligo- and poly-nucleotides in situ on a substrate bycovalently bonding photoremovable groups to the surface of thesubstrate. Selected substrate surface locales are exposed to light toactivate them, by use of a mask. An amino acid or nucleotide monomerwith a photoremovable group is then attached to the activated region.The steps of activation and attachment are repeated to makepolynucleotides and polypeptides of desired length and sequence. Othersynthetic techniques, exemplified by U.S. Pat. Nos. 5,700,637 and6,054,270 to Southern et al., teach the use of inkjet printers, whichare also substantially parallel synthesis because the synthetic patternmust be predefined prior to beginning to “print” the pattern. Thesesolid phase synthesis techniques, which involve the sequential couplingof building blocks (e.g., amino acids) to form the compounds ofinterest, cannot readily be used to prepare many inorganic and organiccompounds.

[0011] U.S. Pat. No. 5,985,356 to Schultz et al. teaches combinatorialchemistry techniques in the field of materials science, providingmethods and a device for synthesis and use of an array of diversematerials in predefined regions of a substrate. An array of differentmaterials on a substrate is prepared by delivering components of variouscompositions of matter to predefined substrate surface locales. Thissynthetic technique permits many classes of materials to be made bysystematic combinatorial methods. Examples of the types of materialsinclude, but are not limited to, inorganic materials, including ionicand covalent crystalline materials, intermetallic materials, metalalloys and composite materials including ceramics. Such materials can bescreened for useful bulk and surface properties as the synthesizedarray, for example, electrical properties, including super- andsemi-conductivity, and thermal, mechanical, thermoelectric, optical,optoelectronic, fluorescent and/or biological properties, includingimmunogenicity.

[0012] Discovery and characterization of materials often requirescombinatorial deposition onto substrates of thin films of preciselyknown chemical composition, concentration, stoichiometry, area and/orthickness. Devices and methods for making arrays of different materials,each with differing composition, concentration, stoichiometry andthin-layer thickness at known substrate locales, permitting systematiccombinatorial array based synthesis and analysis that utilize thin layerdeposition methods, are already known. Although existing thin-layermethods have effected the precision of reagent delivery required to makearrays of different materials, the predefinition required in thesesynthetic techniques is inflexible, and the techniques are slow and thusrelatively costly. Additionally, thin-layer techniques are inherentlyless suited to creating experimental materials under conditions thatdeviate drastically from conditions that are thermodynamicallyreversible or nearly so. Thus, a need exists for more efficient andrapid delivery of precise amounts of reagents needed for materials arraypreparation, with more flexibility as to predetermination and conditionsof formation than attainable by thin-layer methods.

[0013] In combinatorial synthesis of biomacromolecules, U.S. Pat. Nos.5,700,637 and 6,054,270 to Southern et al., as noted previously,describe a method for generating an array of oligonucleotides of chosenmonomeric unit length within discrete cells or regions of a supportmaterial. The in situ method generally described for oligo- orpolynucleotide synthesis involves: coupling a nucleotide precursor to adiscrete predetermined set of cell locations or regions; coupling anucleotide precursor to a second set of cell locations or regions;coupling a nucleotide precursor to a third set of cell locations orregions; and continuing the sequence of coupling steps until the desiredarray has been generated.

[0014] Covalent linking is effected at each location either to thesurface of the support or to a nucleotide coupled in a previous step.

[0015] The patent '637 and patent '270 also teach that impermeablesubstrates are preferable to permeable substrates, such as paper, foreffecting high combinatorial site densities, because the fluid volumesrequired will result in migration or wicking through a permeablesubstrate, precluding attainment of the small feature sizes required forhigh densities (such as those that are attainable by parallelphotolithographic synthesis, which requires a substrate that isoptically smooth and generally also impermeable; see U.S. Pat. No.5,744,305 to Fodor et al.). As the inkjet printing method is a parallelsynthesis technique that requires the array to be “predetermined” innature, and therefore inflexible, and does not enable feature sites inthe micron range or smaller, there remains a need in the art for anon-photolithographic in situ combinatorial array preparation methodthat can provide the high densities attainable by photolithographicarrays, a feat that requires small volumes of reagents and a highlyaccurate deposition method, without the inflexibility of a highlyparallel process that requires a predetermined site sequence. Also, aspermeable substrates offer a greater surface area for localization ofarray constituents, a method of effecting combinatorial high densityarrays non-photolithographically by delivery of sufficiently smallvolumes to permit use of permeable substrates is also an advance overthe current state of the art of array making.

[0016] As explained above, the parallel photolithographic in situformation of biomolecular arrays of high density, e.g., oligonucleotideor polynucleotide arrays, is also known in the art. For example, U.S.Pat. Nos. 5,744,305 and 5,445,934 to Fodor et al. describe arrays ofoligonucleotides and polynucleotides attached to a surface of a planarnon-porous solid support at a density exceeding 400 and 1000 differentoligonucleotides/cm² respectively. The arrays are generated usinglight-directed, spatially addressable synthesis techniques (see alsoU.S. Pat. Nos. 5,143,854 and 5,405,783, and International PatentPublication No. WO 90/15070). With respect to these photolithographicparallel in situ synthesized microarrays, Fodor et al. have developedphotolabile nucleoside and peptide protecting groups, and masking andautomation techniques; see U.S. Pat. No. 5,489,678 and InternationalPatent Publication No. WO 92/10092).

[0017] The aforementioned patents disclose that photolithographictechniques commonly used in semiconductor fabrication may be applied inthe fabrication of arrays of high density. Photolithographic in situsynthesis is best for parallel synthesis, requiring an inordinate numberof masking steps to effect a sequential in situ combinatorial arraysynthesis. Even the parallel combinatorial array synthesis employing aminimized number of masking steps employs a significant number of suchsteps, which increases for each monomeric unit added in the synthesis.Further, the parallel photolithographic in situ array synthesis isinflexible and requires a predetermined mask sequence.

[0018] Because photolithographic fabrication requires a large number ofmasking steps, the yield for this process is lowered relative to anon-photolithographic in situ synthesis by the failure to block and/orinappropriate photo-deblocking by some of the photolabile protectivegroups. These problems with photolabile protective groups compound thepractical yield problem for multi-step in situ syntheses in general byadding photochemical steps to the synthetic process. The problems havenot been addressed by the advances made in the art of making and usingsuch photolabile blockers for in situ synthesis, in part because somephotolabile blocking groups are shielded from the light or “buried” bythe polymer on which they reside, an effect exacerbated with increasingpolymer length. Therefore, the purity of the desired product is low, asthe array will contain significant impurities of undesired products thatcan reduce both sensitivity and selectivity.

[0019] As the photolithographic process for in situ synthesis definessite edges with mask lines, mask imperfections and misalignment,diffractive effects and perturbations of the optical smoothness of thesubstrate can combine to reduce purity by generating polymers similar insequence and/or structure to the desired polymer as impurities, aproblem that becomes more pronounced at the site edges. This isexacerbated when photolithographic protocols attempt to maximize sitedensity by creating arrays that have abutting sites. Because thelikelihood of a mask imperfection or misalignment increases with thenumber of masking steps and the associated number of masks, these edgeeffects are worsened by an increased number of masking steps andutilization of more mask patterns to fabricate a particular array. Siteimpurity, i.e., generation of polymers similar in sequence and/orstructure to the desired polymer, leads to reduced sensitivity andselectivity for arrays designed to analyze a nucleotide sequence.

[0020] Some efforts have been directed to adapting printingtechnologies, particularly, inkjet printing technologies, to formbiomolecular arrays. For example, U.S. Pat. No. 6,015,880 toBaldeschwieler et al. is directed to array preparation using a multistepin situ synthesis. A liquid microdrop containing a first reagent isapplied by a single jet of a multiple jet reagent dispenser to a locuson the surface chemically prepared to permit covalent attachment of thereagent. The reagent dispenser is then displaced relative to thesurface, or the surface is displaced with respect to the dispenser, andat least one microdrop containing either the first reagent or a secondreagent from another dispenser jet is applied to a second substratelocale, which is also chemically activated to be reactive for covalentattachment of the second reagent. Optionally, the second step isrepeated using either the first or second reagents, or differentliquid-borne reagents from different dispenser jets, wherein eachreagent covalently attaches to the substrate surface. The patentdiscloses that inkjet technology may be used to apply the microdrops.

[0021] Ordinary inkjet technology, however, suffers from a number ofdrawbacks. Often, inkjet technology involves heating or using apiezoelectric element to force a fluid through a nozzle in order todirect the ejected fluid onto a surface. Thus, the fluid may be exposedto a surface exceeding 200° C. before being ejected, and most, if notall, peptidic molecules, including proteins, degrade under such extremetemperatures. In addition, forcing peptidic molecules through nozzlescreates shear forces that can alter molecular structure. Nozzles aresubject to clogging, especially when used to eject amacromolecule-containing fluid, and the use of elevated temperaturesexacerbates the problem because liquid evaporation results in depositionof precipitated solids on the nozzles. Clogged nozzles, in turn, canresult in misdirected fluid or ejection of improperly sized droplets.Finally, ordinary inkjet technology employing a nozzle for fluidejection generally cannot be used to deposit arrays with featuredensities comparable to those obtainable using photolithography or othertechniques commonly used in semiconductor processing.

[0022] A number of patents have described the use of acoustic energy inprinting. For example, U.S. Pat. No. 4,308,547 to Lovelady et al.describes a liquid drop emitter that utilizes acoustic principles inejecting droplets from a body of liquid onto a moving document to formcharacters or bar codes thereon. A nozzleless inkjet printing apparatusis used wherein controlled drops of ink are propelled by an acousticalforce produced by a curved transducer at or below the surface of theink. In contrast to inkjet printing devices, nozzleless fluid ejectiondevices described in the aforementioned patent are not subject toclogging and the disadvantages associated therewith, e.g., misdirectedfluid or improperly sized droplets.

[0023] The applicability of nozzleless fluid ejection has generally beenappreciated for ink printing applications. Development of ink printingapplications is primarily economically driven by printing cost and speedfor acceptable text. For acoustic printing, development efforts havetherefore focused on reducing printing costs rather than improvingquality, and on increasing printing speed rather than accuracy. Forexample, U.S. Pat. No. 5,087,931 to Rawson is directed to a system fortransporting ink under constant flow to an acoustic ink printer having aplurality of ejectors aligned along an axis, each ejector associatedwith a free surface of liquid ink. When a plurality of ejectors is usedinstead of a single ejector, printing speed generally increases, butcontrolling fluid ejection, specifically droplet placement, becomes moredifficult.

[0024] U.S. Pat. No. 4,797,693 to Quate describes an acoustic inkprinter for printing polychromatic images on a recording medium. Theprinter is described as comprising a combination of a carrier containinga plurality of differently colored liquid inks, a single acousticprinthead acoustically coupled to the carrier for launching convergingacoustic waves into the carrier, an ink transport means to position thecarrier to sequentially align the differently colored inks with theprinthead, and a controller to modulate the radiation pressure used toeject ink droplets. This printer is described as designed for therealization of cost savings. Because two droplets of primary color,e.g., cyan and yellow, deposited in sufficient proximity will appear asa composite or secondary color, the level of accuracy required is fairlylow and inadequate for biomolecular array formation. Such a printer isparticularly unsuitable for in situ synthesis requiring precise dropletdeposition and consistent placement, so that the proper chemicalreactions occur. That is, the drop placement accuracy needed to effectperception of a composite secondary color is much lower than is requiredfor chemical synthesis at photolithographic density levels.Consequently, an acoustic printing device that is suitable for printingvisually ascertainable material is inadequate for microarraypreparation. Also, this device can eject only a limited quantity of inkfrom the carrier before the liquid meniscus moves out of acoustic focusand drop ejection ceases. This is a significant limitation withbiological fluids, which are typically far more costly and rare thanink. The Quate et al. patent does not address how to use most of thefluid in a closed reservoir without adding additional liquid from anexternal source.

[0025] Thus, there is a general need in the art for improved arraypreparation methodology. An ideal array preparation technique wouldprovide for highly accurate deposition of minute volumes of fluids on asubstrate surface, wherein droplet volume—and thus “spot” size on thesubstrate surface—can be carefully controlled and droplets can beprecisely directed to particular sites on a substrate surface. It wouldalso be optimal if such a technique could be used with porous or evenpermeable surfaces, as such surfaces can provide substantially greatersurface area on which to attach molecular moieties that serve as arrayelements, and would enable preparation of higher density arrays. Todate, as alluded to above, high density arrays have been prepared onlyon nonporous, impermeable surfaces, and only low density arrays could beprepared on porous surfaces.

SUMMARY OF THE INVENTION

[0026] Accordingly, it is an object of the present invention to providemethods and molecular arrays that address the aforementioned need in theart. It has now been discovered that focused acoustic energy can beadvantageously used with very small fluid volumes, on the order of 1picoliter or less, to prepare high density arrays on substrates having aporous or even a permeable surface. Prior array fabrication methods havenot enabled preparation of high density arrays on porous or permeablesurfaces because prior spotting processes are nowhere near as accurateas the present acoustic deposition method, and prior processes have alsorequired larger droplet volumes. In contrast to prior methods ofmanufacturing arrays, then, the present acoustic ejection processenables highly accurate deposition of extremely small liquid droplets,such that diffusion of a deposited droplet into neighboring cells is nota problem, and ultra-high array densities can now be achieved with highporosity, permeable surfaces. Porous, permeable surfaces can providesubstantially more surface area on which to attach molecules within anarray, as the biomolecules can penetrate the surface of the substrateand can thus generate a substantially larger signal per unit ofprojected area due to the resulting non-planar distribution of themolecules on and within the substrate surface.

[0027] The present invention overcomes a significant disadvantage of theprior art, since prior methods could not constrain the formation ordeposition of biomolecules to a small region, due to the inability tolocalize the synthesis process or to restrict the migration of materialsto prevent adjacent “spots” from diffusing into each other. The accurateplacement of extremely small volumes of fluids can enable both multistepin situ synthesis on a substrate surface and deposition of an intactmolecule within an extraordinarily small zone without the need to mask,pre-activate surface sites, or otherwise modify a predetermined regionof a surface.

[0028] The invention makes use of a focused acoustic energy device asdescribed in U.S. patent application Ser. No. U09/669,996 (“AcousticEjection of Fluids from a Plurality of Reservoirs”), inventors Ellson,Foote and Mutz, filed on Sep. 25, 2000, and assigned to Picoliter, Inc.(Mountain View, Calif.). As described in the aforementioned patentapplication, the device enables acoustic ejection of a plurality offluid droplets toward designated sites on a substrate surface fordeposition thereon, and comprises: a plurality of reservoirs or otherfluid-containing means, each adapted to contain a fluid; an acousticejector that includes an acoustic radiation generator and a focusingmeans for focusing the generated acoustic radiation at a focal pointsufficiently near the fluid surface in each of the reservoirs such thatdroplets are ejected therefrom; and a means for positioning the ejectorin acoustic coupling relationship to each of the reservoirs. Preferably,each of the reservoirs is removable, comprised of an individual well ina well plate, and/or arranged in an array. In addition, it is preferredthat the reservoirs are substantially acoustically indistinguishablefrom one another.

[0029] Focused acoustic ejection is carried out by positioning theacoustic ejector so as to be in acoustically coupled relationship with afirst fluid-containing reservoir, and then activating the ejector togenerate and direct acoustic radiation into the fluid so as to eject afluid droplet toward a site on a porous substrate surface. Then, theejector is repositioned so as to be in acoustically coupled relationshipwith a second fluid-containing reservoir and activated again as above toeject a droplet of the second fluid toward a second site on the poroussurface. The method may be repeated with a plurality of fluid reservoirseach containing a fluid, with each reservoir generally although notnecessarily containing a different fluid. The acoustic ejector is thusrepeatedly repositioned so as to eject a droplet from each reservoirtoward a different site on a substrate surface, or toward sites thatalready have a droplet “spot” thereon. In such a way, the method isreadily adapted for use in generating an array of molecular moieties ona porous surface.

[0030] The invention also provides high density arrays of variousmolecular moieties, typically biomolecules, on a porous substratesurface. The arrays provided by the instant invention do not possess theedge effects that result from optical and alignment effects ofphotolithographic masking, nor are they subject to imperfect spottingalignment from inkjet nozzle-directed deposition of reagents. Focusedacoustic ejection can be used to prepare molecular arrays on poroussurfaces that have a density of greater than about 62,500 molecularmoieties, or array elements, per square centimeter of substrate surface,preferably a density of greater than about 250,000, more preferablygreater than about 1,000,000, still more preferably greater than about1,500,000, and most preferably in the range of about 1,500,000 to about4,000,000 molecular moieties per square centimeter of substrate surface.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIGS. 1A and 1B, collectively referred to as FIG. 1, schematicallyillustrate in simplified cross-sectional view an embodiment of theinventive device comprising first and second reservoirs, an acousticejector, and an ejector positioning means. FIG. 1A shows the acousticejector acoustically coupled to the first reservoir and having beenactivated in order to eject a droplet of fluid from within the firstreservoir toward a site on a substrate surface. FIG. 1B shows theacoustic ejector acoustically coupled to a second reservoir.

[0032]FIGS. 2A, 2B, 2C and 2D, collectively referred to as FIG. 2,schematically illustrate in simplified cross-sectional view anembodiment of the inventive method in which a dimer is synthesized insitu on a substrate using the device of FIG. 1. FIG. 2A illustrates theejection of a droplet of surface modification fluid onto a site of asubstrate surface. FIG. 2B illustrates the ejection of a droplet of afirst fluid containing a first molecular moiety adapted for attachmentto the modified surface of the substrate. FIG. 2C illustrates theejection of a droplet of second fluid containing a second molecularmoiety adapted for attachment to the first molecule. FIG. 2D illustratesthe substrate and the dimer synthesized in situ by the processillustrated in FIGS. 2A, 2B and 2C.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific fluids,biomolecules or device structures, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

[0034] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a reservoir” includes a plurality of reservoirs,reference to “a fluid” includes a plurality of fluids, reference to “abiomolecule” includes a combination of biomolecules, and the like.

[0035] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0036] The terms “acoustic coupling” and “acoustically coupled” usedherein refer to a state wherein an object is placed in direct orindirect contact with another object so as to allow acoustic radiationto be transferred between the objects without substantial loss ofacoustic energy. When two entities are indirectly acoustically coupled,an “acoustic coupling medium” is needed to provide an intermediarythrough which acoustic radiation may be transmitted. Thus, an ejectormay be acoustically coupled to a fluid, e.g., by immersing the ejectorin the fluid or by interposing an acoustic coupling medium between theejector and the fluid to transfer acoustic radiation generated by theejector through the acoustic coupling medium and into the fluid.

[0037] The term “adsorb” as used herein refers to the noncovalentretention of a molecule by a substrate surface. That is, adsorptionoccurs as a result of noncovalent interaction between a substratesurface and adsorbing moieties present on the molecule that is adsorbed.Adsorption may occur through hydrogen bonding, van der Waal's forces,polar attraction or electrostatic forces (i.e., through ionic bonding).Examples of adsorbing moieties include, but are not limited to, aminegroups, carboxylic acid moieties, hydroxyl groups, nitroso groups,sulfones and the like. Often the substrate may be functionalized withadsorbent moieties to interact in a certain manner, as when the surfaceis functionalized with amino groups to render it positively charged in apH neutral aqueous environment. Likewise, adsorbate moieties may beadded in some cases to effect adsorption, as when a basic protein isfused with an acidic peptide sequence to render adsorbate moieties thatcan interact electrostatically with a positively charged adsorbentmoiety.

[0038] The term “attached,” as in, for example, a substrate surfacehaving a moiety “attached” thereto, includes covalent binding,adsorption, and physical immobilization. The terms “binding” and “bound”are identical in meaning to the term “attached.”

[0039] The term “array” used herein refers to a two-dimensionalarrangement of features such as an arrangement of reservoirs (e.g.,wells in a well plate) or an arrangement of different materialsincluding ionic, metallic or covalent crystalline, including molecularcrystalline, composite or ceramic, glassine, amorphous, fluidic ormolecular materials on a substrate surface (as in an oligonucleotide orpeptidic array). Different materials in the context of molecularmaterials includes chemical isomers, including constitutional, geometricand stereoisomers, and in the context of polymeric moleculesconstitutional isomers having different monomer sequences. Arrays aregenerally comprised of regular, ordered features, as in, for example, arectilinear grid, parallel stripes, spirals, and the like, butnon-ordered arrays may be advantageously used as well. The arrays orpatterns formed using the devices and methods of the invention generallyhave no optical significance to the unaided human eye. For example, theinvention does not involve ink printing on paper or other substrates inorder to form letters, numbers, bar codes, figures, or otherinscriptions that have optical significance to the unaided human eye. Inaddition, arrays and patterns formed by the deposition of ejecteddroplets on a porous surface as provided herein are preferablysubstantially invisible to the unaided human eye.

[0040] The terms “biomolecule” and “biological molecule” are usedinterchangeably herein to refer to any organic molecule, whethernaturally occurring, recombinantly produced, or chemically synthesizedin whole or in part, that is, was or can be a part of a living organism.The terms encompass, for example, nucleotides, amino acids andmonosaccharides, as well as oligomeric and polymeric species such asoligonucleotides and polynucleotides, peptidic molecules such asoligopeptides, polypeptides and proteins, saccharides such asdisaccharides, oligosaccharides, polysaccharides, mucopolysaccharides orpeptidoglycans (peptido-polysaccharides) and the like. The term alsoencompasses ribosomes, enzyme cofactors, pharmacologically activeagents, and the like.

[0041] The terms “library” and “combinatorial library” are usedinterchangeably herein to refer to a plurality of chemical or biologicalmoieties present on the surface of a substrate, wherein each moiety isdifferent from each other moiety. The moieties may be, e.g., peptidicmolecules and/or oligonucleotides.

[0042] The term “moiety” refers to any particular composition of matter,e.g., a molecular fragment, an intact molecule (including a monomericmolecule, an oligomeric molecule, and a polymer), or a mixture ofmaterials (for example, an alloy or a laminate).

[0043] It will be appreciated that, as used herein, the terms“nucleoside” and “nucleotide” refer to nucleosides and nucleotidescontaining not only the conventional purine and pyrimidine bases, i.e.,adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U), butalso protected forms thereof, e.g., wherein the base is protected with aprotecting group such as acetyl, difluoroacetyl, trifluoroacetyl,isobutyryl or benzoyl, and purine and pyrimidine analogs. Suitableanalogs will be known to those skilled in the art and are described inthe pertinent texts and literature. Common analogs include, but are notlimited to, 1-methyladenine, 2-methyladenine, N⁶-methyladenine,N⁶-isopentyladenine, 2-methylthio-N⁶-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine. In addition, the terms “nucleoside” and “nucleotide”include those moieties that contain not only conventional ribose anddeoxyribose sugars, but other sugars as well. Modified nucleosides ornucleotides also include modifications on the sugar moiety, e.g.,wherein one or more of the hydroxyl groups are replaced with halogenatoms or aliphatic groups, or are functionalized as ethers, amines, orthe like.

[0044] As used herein, the term “oligonucleotide” shall be generic topolydeoxynucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide that is an N-glycoside of a purine or pyrimidine base,and to other polymers containing nonnucleotidic backbones (for examplePNAs), providing that the polymers contain nucleobases in aconfiguration that allows for base pairing and base stacking, such as isfound in DNA and RNA. Thus, these terms include known types ofoligonucleotide modifications, for example, substitution of one or moreof the naturally occurring nucleotides with an analog, inter-nucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.), with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.). There is no intended distinction in length between theterm “polynucleotide” and “oligonucleotide,” and these terms will beused interchangeably. These terms refer only to the primary structure ofthe molecule. As used herein the symbols for nucleotides andpolynucleotides are according to the IUPAC-IUB Commission of BiochemicalNomenclature recommendations (Biochemistry 9:4022, 1970).

[0045] The terms “peptide,” “peptidyl” and “peptidic” as used throughoutthe specification and claims are intended to include any structurecomprised of two or more amino acids. For the most part, the peptides inthe present arrays comprise about 5 to 10,000 amino acids, preferablyabout 5 to 1000 amino acids. The amino acids forming all or a part of apeptide may be any of the twenty conventional, naturally occurring aminoacids, i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid(E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I),lysine (K), leucine (L), methionine (M), asparagine (N), proline (P),glutamine (Q), arginine (R), serine (S), threonine (T), valine (V),tryptophan (W), and tyrosine (Y). Any of the amino acids in the peptidicmolecules forming the present arrays may be replaced by anon-conventional amino acid. In general, conservative replacements arepreferred. Conservative replacements substitute the original amino acidwith a non-conventional amino acid that resembles the original in one ormore of its characteristic properties (e.g., charge, hydrophobicity,stearic bulk; for example, one may replace Val with Nval). The term“non-conventional amino acid” refers to amino acids other thanconventional amino acids, and include, for example, isomers andmodifications of the conventional amino acids (e.g., D-amino acids),non-protein amino acids, post-translationally modified amino acids,enzymatically modified amino acids, constructs or structures designed tomimic amino acids (e.g., α,α-disubstituted amino acids, N-alkyl aminoacids, lactic acid, β-alanine, naphthylalanine, 3-pyridylalanine,4-hydroxyproline, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, and nor-leucine), and peptideshaving the naturally occurring amide —CONH— linkage replaced at one ormore sites within the peptide backbone with a non-conventional linkagesuch as N-substituted amide, ester, thioamide, retropeptide (—NHCO—),retrothioamide (—NHCS—), sulfonamido (—SO₂NH—), and/or peptoid(N-substituted glycine) linkages. Accordingly, the peptidic molecules ofthe array include pseudopeptides and peptidomimetics. The peptides ofthis invention can be (a) naturally occurring, (b) produced by chemicalsynthesis, (c) produced by recombinant DNA technology, (d) produced bybiochemical or enzymatic fragmentation of larger molecules, (e) producedby methods resulting from a combination of methods (a) through (d)listed above, or (f) produced by any other means for producing peptides.

[0046] Peptidic compounds include any pharmacologically active peptide,polypeptide or protein, such as, but not limited to enzymes, monoclonaland polyclonal antibodies, antigens, coagulation modulators, cytokines,endorphins, peptidyl hormones, kinins, and structurally similarbioactive equivalents thereof. By a “structurally similar bioactiveequivalent” is meant a peptidyl compound with structure sufficientlysimilar to that of an identified bioactive peptidyl compound to producesubstantially equivalent therapeutic effects. As used herein and in theappended claims, the terms “protein”, “peptide” and “polypeptide” referto both the specific peptidic compound(s) identified as well asstructurally similar bioactive equivalents thereof.

[0047] Examples of various peptidyl compounds include, but are notlimited to, the following:

[0048] coagulation modulators, such as α₁-antitrypsin, α₂-macroglobulin,antithrombin III, factor I (fibrinogen), factor II (prothrombin), factorIII (tissue prothrombin), factor V (proaccelerin), factor VII(proconvertin), factor VIII (antihemophilic globulin or AHG), factor IX(Christmas factor, plasma thromboplastin component or PTC), factor X(Stuart-Power factor), factor XI (plasma thromboplastin antecedent orPTA), factor XII (Hageman factor), heparin cofactor II, kallikrein,plasmin, plasminogen, prekallikrein, protein C, protein S,thrombomodulin and combinations thereof;.

[0049] cytokines, such as: transforming growth factors (TGFs), includingTGF-β2, TGF-β2, and TGF-β3; bone morphogenetic proteins (for example,BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);heparin-binding growth factors (for example, fibroblast growth factor(FGF), epidermal growth factor (EGF), platelet-derived growth factor(PDGF), heparin-binding neurotrophic factor (HBNF), and insulin-likegrowth factor (IGF)); connective tissue activated peptides (CTAPs),osteogenic factors; colony stimulating factor; interferons, includinginterferon-α, interferon α-2a, interferon α-2b, interferon α-n3,interferon-β, and interferon-γ; interleukins, including interleukin-1,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin-12, interleukin-13,interleukin-14, interleukin-15, interleukin-16, and interleukin-17;tumor necrosis factor; tumor necrosis factor-α; granuloyctecolony-stimulating factor (G-CSF); granulocyte-macrophagecolony-stimulating factor (GM-CSF); macrophage colony-stimulatingfactor; Inhibins (e.g., Inhibin A and Inhibin B); growth differentiatingfactors (e.g., GDF-1); Activins (e.g., Activin A, Activin B, and ActivinAB); midkine (MD); and thymopoietin.

[0050] endorphins, i.e., peptides that activate opiate receptors,including pharmacologically active endorphin derivatives such asdermorphin, dynorphin, α-endorphin, β-endorphin, γ-endorphin,σ-endorphin [Leu⁵]enkephalin, [Met⁵]enkephalin, substance P, andcombinations thereof;

[0051] peptidyl hormones, such as activin, amylin, angiotensin, atrialnatriuretic peptide (ANP), calcitonin (derived from chicken, eel, human,pig, rat, salmon, etc.), calcitonin gene-related peptide, calcitoninN-terminal flanking peptide, cholecystokinin (CCK), ciliary neurotrophicfactor (CNTF), corticotropin (adrenocorticotropin hormone, ACTH),corticotropin-releasing factor (CRF or CRH), follicle-stimulatinghormone (FSH), gastrin, gastrin inhibitory peptide (GIP),gastrin-releasing peptide, glucagon, gonadotropin-releasing factor (GnRFor GNRH), growth hormone releasing factor (GRF, GRH), human chorionicgonadotropin (hCH), inhibin A, inhibin B, insulin (derived from beef,human, pig, etc.), leptin, lipotropin (LPH), luteinizing hormone (LH),luteinizing hormone-releasing hormone (LHRH), lypressin,α-melanocyte-stimulating hormone, β-melanocyte-stimulating hormone,γ-melanocyte-stimulating hormone, melatonin, motilin, oxytocin(pitocin), pancreatic polypeptide, parathyroid hormone (PTH), placentallactogen, prolactin (PRL), prolactin-release inhibiting factor (PIF),prolactin-releasing factor (PRF), secretin, somatostatin, somatotropin(growth hormone, GH), somatostatin (SIF, growth hormone-releaseinhibiting factor, GIF), thyrotropin (thyroid-stimulating hormone, TSH),thyrotropin-releasing factor (TRH or TRF), thyroxine, triiodothyronine,vasoactive intestinal peptide (VIP), and vasopressin (antidiuretichormone, ADH);.

[0052] analogues of LHRH, such as buserelin, deslorelin, fertirelin,goserelin, histrelin, leuprolide (leuprorelin), lutrelin, nafarelin,tryptorelin and combinations thereof;

[0053] kinins, such as bradykinin, potentiator B, bradykinin potentiatorC, and kallidin and combinations thereof.

[0054] enzymes, such as transferases, hydrolases, isomerases, proteases,ligases and oxidoreductases such as esterases, phosphatases,glycosidases and peptidases. Specific examples of enzymes include superoxide dismutase (SOD), tissue plasminogen activator (TPA), renin,adenosine deaminase, β-glucocerebrosidase, asparaginase, dornase-α,hyaluronidase, elastase, trypsin, thymidin kinase (TK), tryptophanhydroxylase, urokinase, and kallikrein;

[0055] enzyme inhibitors, such as leupeptin, chymostatin, pepslatin,renin inhibitors, angiotensin converting enzyme (ACE) inhibitors, andthe like;

[0056] antibodies, including both monoclonal and polyclonal antibodies,as well as antibody fragments, such as the F(ab′)₂, Fab, Fv and Fcfragments of monoclonal antibodies; and

[0057] other peptidyl drugs, such as abarelix, anakinra, ancestim,bivalirudin, bleomycin, bombesin, desmopressin acetate, des-Q14-ghrelin,enterostatin, erythropoeitin, exendin-4, filgrastim, gonadorelin,insulinotropin, lepirudin, magainin I, magainin II, nerve growth factor,pentigetide, thrombopoietin, thymosin α-1, and urotensin II andcombinations thereof.

[0058] The term “fluid” as used herein refers to matter that is nonsolidor at least partially gaseous and/or liquid. A fluid may contain a solidthat is minimally, partially or fully solvated, dispersed or suspended.Examples of fluids include, without limitation, aqueous liquids(including water per se and salt water) and nonaqueous liquids such asorganic solvents and the like. As used herein, the term “fluid” is notsynonymous with the term “ink” in that an ink must contain a colorantand may not be gaseous.

[0059] The term “near” is used to refer to the distance from the focalpoint of the focused acoustic radiation to the surface of the fluid fromwhich a droplet is to be ejected. The distance should be such that thefocused acoustic radiation directed into the fluid results in dropletejection from the fluid surface, and one of ordinary skill in the artwill be able to select an appropriate distance for any given fluid usingstraightforward and routine experimentation. Generally, however, asuitable distance between the focal point of the acoustic radiation andthe fluid surface is in the range of about 1 to about 15 times thewavelength of the speed of sound in the fluid, more typically in therange of about 1 to about 10 times that wavelength, preferably in therange of about 1 to about 5 times that wavelength.

[0060] The terms “focusing means” and “acoustic focusing means” refer toa means for causing acoustic waves to converge at a focal point byeither a device separate from the acoustic energy source that acts likean optical lens, or by the spatial arrangement of acoustic energysources to effect convergence of acoustic energy at a focal point byconstructive and destructive interference. A focusing means may be assimple as a solid member having a curved surface, or it may includecomplex structures such as those found in Fresnel lenses, which employdiffraction in order to direct acoustic radiation. Suitable focusingmeans also include phased array methods as known in the art anddescribed, for example, in U.S. Pat. No. 5,798,779 to Nakayasu et al.and Amemiya et al. (1997) Proceedings of the 1997 IS&T NIP13International Conference on Digital Printing Technologies Proceedings,at pp. 698-702.

[0061] The term “reservoir” as used herein refers to a receptacle orchamber for holding or containing a fluid. Thus, a fluid in a reservoirnecessarily has a free surface, i.e., a surface that allows a droplet tobe ejected therefrom. A reservoir may also be a locus on a substratesurface within which a fluid is constrained.

[0062] The term “substrate” as used herein refers to a substrate havinga porous surface. The entire substrate may be porous, in which case theporous surface is the surface of the substrate itself. Alternatively, anonporous substrate may be used providing that a porous surface layer ispresent that provides the porous surface. The substrate may beconstructed in any of a number of forms such as wafers, slides, wellplates, membranes, etc. Suitable substrates include uncoated porousglass slides, including controlled pore glass (CPG) slides; porous glassslides coated with a polymeric coating, e.g., an aminosilane orpoly-L-lysine coating, thus having a porous polymeric surface; andnonporous glass slides coated with a porous coating, such as may becomprised of a cellulosic polymer (e.g., nitrocellulose),polyacrylamide, or a porous metal (for example, comprised of microporousaluminum). Examples of commercially available substrates having poroussurfaces include the Fluorescent Array Surface Technology (FAST™) slidesavailable from Schleicher & Schuell, Inc. (Keene, N.H.), which arecoated with a 10-30 μm thick porous, fluid-permeable nitrocelluloselayer that substantially increases the available binding area per unitarea of surface. Other commercially available porous substrates includethe CREATIVECHIP® permeable slides currently available from Eppendorf AG(Hamburg, Germany), and substrates having “three-dimensional” geometry,by virtue of an ordered, highly porous structure that enables reagentsto flow into and penetrate through the pores and channels of the entirestructure. Such substrates are available from Gene Logic, Inc. under thetradename “Flow-Thru Chip,” and are described by Steel et al. in Chapter5 of Microarray Biochip Technology (BioTechniques Books, Natick, Mass.,2000).

[0063] The term “porous” as in a “porous substrate” or a “substratehaving a porous surface,” refers to a substrate or surface,respectively, having a porosity (void percentage) in the range of about1% to about 99%, preferably about 5% to about 99%, more preferably inthe range of about 15% to about 95%, and an average pore size of about100 Å to about 1 mm, typically about 500 Å to about 0.5 mm.

[0064] The term “impermeable” is used in the conventional sense to meannot permitting water or other fluid to pass through. The term“permeable” as used herein means not “impermeable.” Thus, a “permeablesubstrate” and a “substrate having a permeable surface” refer to asubstrate or surface, respectively, which can be permeated with water orother fluid.

[0065] While the foregoing support materials are representative ofconventionally used substrates, it is to be understood that a substratemay in fact comprise any biological, nonbiological, organic and/orinorganic material so long as a porous surface is provided, and may bein any of a variety of physical forms, e.g., particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, and the like, and may further have anydesired shape, such as a disc, square, sphere, circle, etc. Thesubstrate surface may or may not be flat, e.g., the surface may containraised or depressed regions. A substrate may additionally contain or bederivatized to contain reactive functionalities that covalently link acompound to the substrate surface. These are widely known and include,for example, silicon dioxide supports containing reactive Si—OH groups,polyacrylamide supports, polystyrene supports, polyethylene glycolsupports, and the like.

[0066] The term “surface modification” as used herein refers to thechemical and/or physical alteration of a surface by an additive orsubtractive process to change one or more chemical and/or physicalproperties of a substrate surface or a selected site or region of asubstrate surface. For example, surface modification may involve (1)changing the wetting properties of a surface, (2) functionalizing asurface, i.e., providing, modifying or substituting surface functionalgroups, (3) defunctionalizing a surface, i.e., removing surfacefunctional groups, (4) otherwise altering the chemical composition of asurface, e.g., through etching, (5) increasing or decreasing surfaceroughness, (6) providing a coating on a surface, e.g., a coating thatexhibits wetting properties that are different from the wettingproperties of the surface, and/or (7) depositing particulates on asurface.

[0067] “Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

[0068] The term “substantially” as in, for example, the phrase“substantially all molecules of an array,” refers to at least 90%,preferably at least 95%, more preferably at least 99%, and mostpreferably at least 99.9%, of the molecules of an array. Other uses ofthe term “substantially” involve an analogous definition.

[0069] The invention accordingly provides a method for acousticallygenerating fluid droplets from a plurality of individual reservoirs toform a high density molecular array on a porous substrate surface. Thatis, focused acoustic energy is used to eject single fluid droplets fromthe free surface of a fluid (e.g., in a reservoir or well plate),generally toward discrete sites on a porous substrate surface, enablingextraordinarily precise deposition and consistent droplet size andvelocity. The device comprises a plurality of reservoirs, each adaptedto contain a fluid, an ejector comprising an acoustic radiationgenerator for generating acoustic radiation and a focusing means forfocusing the acoustic radiation generated (at a focal point within andsufficiently near the fluid surface in each of the reservoirs to resultin the ejection of droplets therefrom), and a means for positioning theejector in acoustic coupling relationship to each of the reservoirs.

[0070] The use of such a focused acoustic ejection system enablespreparation of arrays on porous surfaces that will generally have adensity in the range of at least 62,500 molecular moieties as array“elements” (e.g., surface-bound oligomers) per square centimeter ofsubstrate surface, preferably at least 250,000 molecular moieties persquare centimeter of substrate surface, more preferably at least1,000,000, most preferably at least 1,500,000, and optimally in therange of about 1,500,000 to about 4,000,000 molecular moieties persquare centimeter of substrate surface. Each molecular moiety may bedifferent from every other molecular moiety within the array, such thatthe array is a “combinatorial” library of the different molecularmoieties.

[0071] A significant advantage of using focused acoustic energytechnology in the manufacture of arrays is that the aforementioned arraydensities may be achieved on substrates with porous surfaces, and evenpermeable surfaces. More specifically, the present acoustic ejectionmethod can be used to manufacture high density arrays that can be readwith a high precision digitizing scanner capable of 2 μm resolution, bydepositing droplets having a volume on the order of 1 pL, resulting indeposited spots about 18 μm in diameter. For ultra-high density arrays,a smaller droplet volume is necessary, typically less than about 0.03 pL(deposition of droplets having a volume on the order of 0.025 pL willresult in deposited spots about 4.5 μm in diameter). Localization ofdeposited droplets using chemical or physical means, such as describedin 6,054,270 to Southern et al., is unnecessary because acousticejection enables precisely directed minute droplets to be deposited withextraordinary accuracy at a particular site.

[0072]FIG. 1 illustrates a focused acoustic ejection device insimplified cross-sectional view. As with all figures referenced herein,in which like parts are referenced by like numerals, FIG. 1 is not toscale, and certain dimensions may be exaggerated for clarity ofpresentation. The device 11 includes a plurality of reservoirs, i.e., atleast two reservoirs, with a first reservoir indicated at 13 and asecond reservoir indicated at 15, each adapted to contain a fluid havinga fluid surface, e.g., a first fluid 14 and a second fluid 16 havingfluid surfaces respectively indicated at 17 and 19. Fluids 14 and 16 maythe same or different. As shown, the reservoirs are of substantiallyidentical construction so as to be substantially acousticallyindistinguishable, but identical construction is not a requirement. Thereservoirs are shown as separate removable components but may, ifdesired, be fixed within a plate or other substrate. For example, theplurality of reservoirs may comprise individual wells in a well plate,optimally although not necessarily arranged in an array. Each of thereservoirs 13 and 15 is preferably axially symmetric as shown, havingvertical walls 21 and 23 extending upward from circular reservoir bases25 and 27 and terminating at openings 29 and 31, respectively, althoughother reservoir shapes may be used. The material and thickness of eachreservoir base should be such that acoustic radiation may be transmittedtherethrough and into the fluid contained within the reservoirs.

[0073] The device also includes an acoustic ejector 33 comprised of anacoustic radiation generator 35 for generating acoustic radiation and afocusing means 37 for focusing the acoustic radiation at a focal pointwithin the fluid from which a droplet is to be ejected, near the fluidsurface. As shown in FIG. 1, the focusing means 37 may comprise a singlesolid piece having a concave surface 39 for focusing acoustic radiation,but the focusing means may be constructed in other ways as discussedbelow. The acoustic ejector 33 is thus adapted to generate and focusacoustic radiation so as to eject a droplet of fluid from each of thefluid surfaces 17 and 19 when acoustically coupled to reservoirs 13 and15 and thus to fluids 14 and 16, respectively. The acoustic radiationgenerator 35 and the focusing means 37 may function as a single unitcontrolled by a single controller, or they may be independentlycontrolled, depending on the desired performance of the device.Typically, single ejector designs are preferred over multiple ejectordesigns because accuracy of droplet placement and consistency in dropletsize and velocity are more easily achieved with a single ejector.

[0074] As will be appreciated by those skilled in the art, any of avariety of focusing means may be employed in conjunction with thepresent invention. For example, one or more curved surfaces may be usedto direct acoustic radiation to a focal point near a fluid surface. Onesuch technique is described in U.S. Pat. No. 4,308,547 to Lovelady etal. Focusing means with a curved surface have been incorporated into theconstruction of commercially available acoustic transducers such asthose manufactured by Panametrics Inc. (Waltham, Mass.). In addition,Fresnel lenses are known in the art for directing acoustic energy at apredetermined focal distance from an object plane. See, e.g., U.S. Pat.No. 5,041,849 to Quate et al. Fresnel lenses may have a radial phaseprofile that diffracts a substantial portion of acoustic energy into apredetermined diffraction order at diffraction angles that vary radiallywith respect to the lens. The diffraction angles should be selected tofocus the acoustic energy within the diffraction order on a desiredobject plane.

[0075] There are also a number of ways to acoustically couple theejector 33 to each individual reservoir and thus to the fluid therein.One such approach is through direct contact as is described, forexample, in U.S. Pat. No. 4,308,547 to Lovelady et al., wherein afocusing means constructed from a hemispherical crystal having segmentedelectrodes is submerged in a liquid to be ejected. The aforementionedpatent further discloses that the focusing means may be positioned at orbelow the surface of the liquid. However, this approach for acousticallycoupling the focusing means to a fluid is undesirable when the ejectoris used to eject different fluids in a plurality of containers orreservoirs, as repeated cleaning of the focusing means would be requiredin order to avoid cross-contamination. The cleaning process wouldnecessarily lengthen the transition time between each droplet ejectionevent. In addition, in such a method, fluid would adhere to the ejectoras it is removed from each container, wasting material that may becostly or rare.

[0076] Thus, a preferred approach would be to acoustically couple theejector to the reservoirs and reservoir fluids without contacting anyportion of the ejector, e.g., the focusing means, with any of the fluidsto be ejected. To this end, the present invention provides an ejectorpositioning means for positioning the ejector in controlled andrepeatable acoustic coupling with each of the fluids in the reservoirsto eject droplets therefrom without submerging the ejector therein. Thistypically involves direct or indirect contact between the ejector andthe external surface of each reservoir. When direct contact is used inorder to acoustically couple the ejector to each reservoir, it ispreferred that the direct contact is wholly conformal to ensureefficient acoustic energy transfer. That is, the ejector and thereservoir should have corresponding surfaces adapted for mating contact.Thus, if acoustic coupling is achieved between the ejector and reservoirthrough the focusing means, it is desirable for the reservoir to have anoutside surface that corresponds to the surface profile of the focusingmeans. Without conformal contact, efficiency and accuracy of acousticenergy transfer may be compromised. In addition, since many focusingmeans have a curved surface, the direct contact approach may necessitatethe use of reservoirs having a specially formed inverse surface.

[0077] Optimally, acoustic coupling is achieved between the ejector andeach of the reservoirs through indirect contact, as illustrated in FIG.1A. In the figure, an acoustic coupling medium 41 is placed between theejector 33 and the base 25 of reservoir 13, with the ejector andreservoir located at a predetermined distance from each other. Theacoustic coupling medium may be an acoustic coupling fluid, preferablyan acoustically homogeneous material in conformal contact with both theacoustic focusing means 37 and each reservoir. In addition, it isimportant to ensure that the fluid medium is substantially free ofmaterial having different acoustic properties than the fluid mediumitself. As shown, the first reservoir 13 is acoustically coupled to theacoustic focusing means 37 such that an acoustic wave is generated bythe acoustic radiation generator and directed by the focusing means 37into the acoustic coupling medium 41, which then transmits the acousticradiation into the reservoir 13.

[0078] In operation, reservoirs 13 and 15 of the device are each filledwith first and second fluids 14 and 16, respectively, as shown inFIG. 1. The acoustic ejector 33 is positionable by means of ejectorpositioning means 43, shown below reservoir 13, in order to achieveacoustic coupling between the ejector and the reservoir through acousticcoupling medium 41. Substrate 45 is positioned above and in proximity tothe first reservoir 13 such that one surface of the substrate, shown inFIG. 1 as underside surface 51, faces the reservoir and is substantiallyparallel to the surface 17 of the fluid 14 therein. Once the ejector,the reservoir and the substrate are in proper alignment, the acousticradiation generator 35 is activated to produce acoustic radiation thatis directed by the focusing means 37 to a focal point 47 near the fluidsurface 17 of the first reservoir. As a result, droplet 49 is ejectedfrom the fluid surface 17 onto a designated site on the undersidesurface 51 of the substrate. The ejected droplet may be retained on thesubstrate surface by solidifying thereon after contact; in such anembodiment, it is necessary to maintain the substrate at a lowtemperature, i.e., a temperature that results in droplet solidificationafter contact. Alternatively, or in addition, a molecular moiety withinthe droplet attaches to the substrate surface after contract, throughadsorption, physical immobilization, or covalent binding.

[0079] Then, as shown in FIG. 1B, a substrate positioning means 50repositions the substrate 45 over reservoir 15 in order to receive adroplet therefrom at a second designated site. FIG. 1B also shows thatthe ejector 33 has been repositioned by the ejector positioning means 43below reservoir 15 and in acoustically coupled relationship thereto byvirtue of acoustic coupling medium 41. Once properly aligned as shown inFIG. 1B, the acoustic radiation generator 35 of ejector 33 is activatedto produce acoustic radiation that is then directed by focusing means 37to a focal point within fluid 16 near the fluid surface 19, therebyejecting droplet 53 onto the substrate. It should be evident that suchoperation is illustrative of how the inventive device may be used toeject a plurality of fluids from reservoirs in order to form a pattern,e.g., an array, on the substrate surface 51. It should be similarlyevident that the device may be adapted to eject a plurality of dropletsfrom one or more reservoirs onto the same site of the substrate surface.

[0080] As discussed above, either individual, e.g., removable,reservoirs or well plates may be used to contain fluids that are to beejected, wherein the reservoirs or the wells of the well plate arepreferably substantially acoustically indistinguishable from oneanother. Also, unless it is intended that the ejector is to be submergedin the fluid to be ejected, the reservoirs or well plates must haveacoustic transmission properties sufficient to allow acoustic radiationfrom the ejector to be conveyed to the surfaces of the fluids to beejected. Typically, this involves providing reservoir or well bases thatare sufficiently thin to allow acoustic radiation to travel therethroughwithout unacceptable dissipation. In addition, the material used in theconstruction of reservoirs must be compatible with the fluids containedtherein. Thus, if it is intended that the reservoirs or wells contain anorganic solvent such as acetonitrile, polymers that dissolve or swell inacetonitrile would be unsuitable for use in forming the reservoirs orwell plates. For water-based fluids, a number of materials are suitablefor the construction of reservoirs and include, but are not limited to,ceramics such as silicon oxide and aluminum oxide, metals such asstainless steel and platinum, and polymers such as polyester andpolytetrafluoroethylene. Many well plates suitable for use with theemployed device are commercially available and may contain, for example,96, 384 or 1536 wells per well plate. Manufactures of suitable wellplates for use in the employed device include Corning Inc. (Coming,N.Y.) and Greiner America, Inc. (Lake Mary, Fla.). However, theavailability of such commercially available well plates does notpreclude manufacture and use of custom-made well plates containing atleast about 10,000 wells, or as many as 100,000 wells or more. For arrayforming applications, it is expected that about 100,000 to about4,000,000 or more reservoirs may be employed. In addition, to reduce theamount of movement and time needed to align the ejector with eachreservoir or reservoir well, it is preferable that the center of eachreservoir is located not more than about 1 centimeter, preferably notmore than about 1 millimeter and optimally not more than about 0.5millimeter from a neighboring reservoir center.

[0081] Moreover, the present methodology may be adapted to eject fluidsof virtually any type and amount desired. The fluid may be aqueousand/or nonaqueous. Examples of fluids include, but are not limited to,aqueous fluids such as water per se and water-solvated ionic andnon-ionic solutions, organic solvents, and lipidic liquids, suspensionsof immiscible fluids and suspensions or slurries of solids in liquids.Because the invention is readily adapted for use with high temperatures,fluids such as liquid metals, ceramic materials, and glasses may beused; see, e.g., co-pending patent application U.S. Ser. No. 09/669/194(“Method and Apparatus for Generating Droplets of Immiscible Fluids”),inventors Ellson and Mutz, filed on Sep. 25, 2000, and assigned toPicoliter, Inc. (Mountain View, Calif.). U.S. Pat. Nos. 5,520,715 and5,722,479 to Oeftering describe the use of acoustic ejection for liquidmetal for forming structures using a single reservoir and adding fluidto maintain focus. U.S. Pat. No. 6,007,183 to Horine is another patentthat pertains to the use of acoustic energy to eject droplets of liquidmetal. The capability of producing fine droplets of such materials is insharp contrast to piezoelectric technology, insofar as piezoelectricsystems perform suboptimally at elevated temperatures. Furthermore,because of the precision that is possible using the inventivetechnology, the device may be used to eject droplets from a reservoiradapted to contain no more than about 100 nanoliters of fluid,preferably no more than 10 nanoliters of fluid. In certain cases, theejector may be adapted to eject a droplet from a reservoir adapted tocontain about 1 to about 100 nanoliters of fluid. This is particularlyuseful when the fluid to be ejected contains rare or expensivebiomolecules, wherein it may be desirable to eject droplets having avolume of about 1 picoliter or less, e.g., having a volume in the rangeof about 0.025 pL to about 1 pL.

[0082] It will be appreciated that various components of a suitableacoustic ejection device may require individual control orsynchronization to form an array on a substrate. For example, theejector positioning means may be adapted to eject droplets from eachreservoir in a predetermined sequence associated with an array to beprepared on a substrate surface. Similarly, the substrate positioningmeans for positioning the substrate surface with respect to the ejectormay be adapted to position the substrate surface to receive droplets ina pattern or array thereon. Either or both positioning means, i.e., theejector positioning means and the substrate positioning means, may beconstructed from, for example, motors, levers, pulleys, gears, acombination thereof, or other electromechanical or mechanical meansknown to one of ordinary skill in the art. It is preferable to ensurethat there is a correspondence between the movement of the substrate,the movement of the ejector and the activation of the ejector to ensureproper array formation.

[0083] The device may also include certain performance-enhancingfeatures. For example, the device may include a cooling means forlowering the temperature of the substrate surface to ensure, forexample, that the ejected droplets adhere to the substrate. The coolingmeans may be adapted to maintain the substrate surface at a temperaturethat allows fluid to partially or preferably substantially solidifyafter the fluid comes into contact therewith. In the case of aqueousfluids, the cooling means should have the capacity to maintain thesubstrate surface at about 0° C. In addition, repeated application ofacoustic energy to a reservoir of fluid may result in heating of thefluid. Heating can of course result in unwanted changes in fluidproperties such as viscosity, surface tension and density. Thus, thedevice may further comprise means for maintaining fluid in thereservoirs at a constant temperature. Design and construction of suchtemperature maintaining means are known to one of ordinary skill in theart and may comprise, e.g., components such a heating element, a coolingelement, or a combination thereof. For many biomolecular depositionapplications, it is generally desired that the fluid containing thebiomolecule is kept at a constant temperature without deviating morethan about 1° C. or 2° C. therefrom. In addition, for a biomolecularfluid that is particularly heat sensitive, it is preferred that thefluid be kept at a temperature that does not exceed about 10° C. abovethe melting point of the fluid, preferably at a temperature that doesnot exceed about 5° C. above the melting point of the fluid. Thus, forexample, when the biomolecule-containing fluid is aqueous, it may beoptimal to keep the fluid at about 4° C. during ejection.

[0084] For some applications, especially those involving acousticdeposition of molten metals or other materials, a heating element may beprovided for maintaining the substrate at a temperature below themelting point of the molten material, but above ambient temperature sothat control of the rapidity of cooling may be effected. The rapidity ofcooling may thus be controlled, to permit experimentation regarding theproperties of combinatorial compositions such as molten deposited alloyscooled at different temperatures. For example, it is known thatmetastable materials are generally more likely to be formed with rapidcooling, and other strongly irreversible conditions. The approach ofgenerating materials by different cooling or quenching rates my betermed combinatorial quenching, and could be effected by changing thesubstrate temperature between acoustic ejections of the molten material.A more convenient method of evaluating combinatorial compositionssolidified from the molten state at different rates is by generatingmultiple arrays having the same pattern of nominal compositions ejectedacoustically in the molten state onto substrates maintained at differenttemperatures.

[0085] In some cases, a substrate surface may be modified prior toformation of an array thereon. Surface modification may involvefunctionalization or defunctionalization, smoothing or roughening,changing surface conductivity, coating, degradation, passivation orotherwise altering the surface's chemical composition or physicalproperties. A preferred surface modification method involves alteringthe wetting properties of the surface, for example to facilitateconfinement of a droplet ejected on the surface within a designated areaor enhancement of the kinetics for the surface attachment of molecularmoieties contained in the ejected droplet. A preferred method foraltering the wetting properties of the substrate surface involvesdeposition of droplets of a suitable surface modification fluid at eachdesignated site of the substrate surface prior to acoustic ejection offluids to form an array thereon. In this way, the “spread” of theacoustically ejected droplets may be optimized and consistency in spotsize (i.e., diameter, height and overall shape) ensured. One way toimplement the method involves acoustically coupling the ejector to amodifier reservoir containing a surface modification fluid and thenactivating the ejector, as described in detail above, to produce andeject a droplet of surface modification fluid toward a designated siteon the substrate surface. The method is repeated as desired to depositsurface modification fluid at additional designated sites. This methodis useful in a number of applications including, but not limited to,spotting oligomers to form an array on a substrate surface orsynthesizing array oligomers in situ. As noted above, other physicalproperties of the surface that may be modified include thermalproperties and electrical conductivity.

[0086]FIG. 2 schematically illustrates in simplified cross-sectionalview a specific embodiment of the aforementioned method in which a dimeris synthesized on a substrate using a device similar to that illustratedin FIG. 1, but including a modifier reservoir 59 containing a surfacemodification fluid 60 having a fluid surface 61. FIG. 2A illustrates theejection of a droplet 63 of surface modification fluid 60 selected toalter the wetting properties of a designated site on surface 51 of thesubstrate 45 where the dimer is to be synthesized. The ejector 33 ispositioned by the ejector positioning means 43 below modifier reservoir59 in order to achieve acoustic coupling therewith through acousticcoupling medium 41. Substrate 45 is positioned above the modifierreservoir 19 at a location that enables acoustic deposition of a dropletof surface modification fluid 60 at a designated site. Once the ejector33, the modifier reservoir 59 and the substrate 45 are in properalignment, the acoustic radiation generator 35 is activated to produceacoustic radiation that is directed by the focusing means 37 in a mannerthat enables ejection of droplet 63 of the surface modification fluid 60from the fluid surface 61 onto a designated site on the undersidesurface 51 of the substrate. Once the droplet 63 contacts the substratesurface 51, the droplet modifies an area of the substrate surface toresult in an increase or decrease in the surface energy of the area withrespect to deposited fluids.

[0087] Then, as shown in FIG. 2B, the substrate 45 is repositioned bythe substrate positioning means 50 such that the region of the substratesurface modified by droplet 63 is located directly over reservoir 13.FIG. 3B also shows that the ejector 33 is positioned by the ejectorpositioning means below reservoir 13 to acoustically couple the ejectorand the reservoir through acoustic coupling medium 41. Once properlyaligned, the ejector 33 is again activated so as to eject droplet 49onto substrate. Droplet 49 contains a first monomeric moiety 65,preferably a biomolecule such as a protected nucleoside or amino acid,which after contact with the substrate surface attaches thereto bycovalent bonding or adsorption.

[0088] Then, as shown in FIG. 2C, the substrate 45 is again repositionedby the substrate positioning means 50 such that the site having thefirst monomeric moiety 65 attached thereto is located directly overreservoir 15 in order to receive a droplet therefrom. FIG. 3B also showsthat the ejector 33 is positioned by the ejector positioning means belowreservoir 15 to acoustically couple the ejector and the reservoirthrough acoustic coupling medium 41. Once properly aligned, the ejector33 is again activated so as to eject droplet 53 is ejected ontosubstrate. Droplet 53 contains a second monomeric moiety 67, adapted forattachment to the first monomeric moiety 65, typically involvingformation of a covalent bond so as to generate a dimer as illustrated inFIG. 3D. The aforementioned steps may be repeated to generate anoligomer, e.g., an oligonucleotide, of a desired length.

[0089] The chemistry employed in synthesizing substrate-boundoligonucleotides in this way will generally involve now-conventionaltechniques known to those skilled in the art of nucleic acid chemistryand/or described in the pertinent literature and texts. See, forexample, DNA Microarrays: A Practical Approach, M. Schena, Ed. (OxfordUniversity Press, 1999). That is, the individual coupling reactions areconducted under standard conditions used for the synthesis ofoligonucleotides and conventionally employed with automatedoligonucleotide synthesizers. Such methodology is described, forexample, in D. M. Matteuci et al. (1980) Tet. Lett. 521:719, U.S. Pat.No. 4,500,707 to Caruthers et al., and U.S. Pat. Nos. 5,436,327 and5,700,637 to Southern et al.

[0090] Alternatively, an oligomer may be synthesized prior to attachmentto the substrate surface and then “spotted” onto a particular locus onthe surface using the focused acoustic ejection methodology described indetail above. Again, the oligomer may be an oligonucleotide, anoligopeptide, or any other biomolecular (or nonbiomolecular) oligomermoiety. Preparation of substrate-bound peptidic molecules, e.g., in theformation of peptide arrays and protein arrays, is described inco-pending patent application U.S. Ser. No. 09/669,997 (“FocusedAcoustic Energy in the Preparation of Peptidic Arrays”), inventors Mutzand Ellson, filed Sep. 25, 2000 and assigned to Picoliter, Inc.(Mountain View, Calif.). Preparation of substrate-boundoligonucleotides, particularly arrays of oligonucleotides wherein atleast one of the oligonucleotides contains one or more partiallynonhybridizing segments, is described in co-pending patent applicationU.S. Ser. No. 09/699,267 (“Arrays of Oligonucleotides ContainingNonhybridizing Segments”), inventor Ellson, also filed on Sep. 25, 2000and assigned to Picoliter, Inc. Preparation of other types of arraysusing focused acoustic energy is described in co-pending patentapplication U.S. Ser. No. 09/17271392, filed on Nov. 29, 2000 and alsoassigned to Picoliter, Inc.

[0091] It will be appreciated by those in the art that the invention isuseful in the preparation of high density combinatorial librariescontaining a variety of synthetic, semi-synthetic or naturally occurringmolecular moieties, insofar as focused acoustic energy makes possiblethe use and manipulation of extremely small volumes of fluids withextraordinary accuracy. This is in sharp contrast to prior techniquesfor preparing combinatorial libraries, with which effective spot-to-spotbinding cannot be guaranteed. Furthermore, piezoelectric jettechnologies are limited with respect to the fluids that may be usedsince high temperatures are required, while the present invention doesnot require high temperatures (although heat may be necessary in somecases, i.e., with fluids having high melting points) and thus allows forthe possibility of using fluids that may be heat-sensitive or evenflammable.

[0092] It should be evident, then, that many variations of the inventionare possible. For example, each of the ejected droplets may be depositedas an isolated and “final” feature, e.g., in spotting oligonucleotides,as mentioned above. Alternatively, or in addition, a plurality ofejected droplets may be deposited on the same location of a substratesurface in order to synthesize a biomolecular array in situ, asdescribed above. For array fabrication, it is expected that variouswashing steps may be used between droplet ejection steps. Such washsteps may involve, e.g., submerging the entire substrate surface onwhich features have been deposited in a washing fluid. In a modificationof this process, the substrate surface may be deposited on a fluidcontaining a reagent that chemically alters all features atsubstantially the same time, e.g., to activate and/or deprotectbiomolecular features already deposited on the substrate surface toprovide sites on which additional coupling reactions may occur.

[0093] The focused acoustic energy methodology described herein enablesejection of droplets at a rate of at least about 1,000,000 droplets perminute from the same reservoir, and at a rate of at least about 100,000drops per minute from different reservoirs. In addition, currentpositioning technology allows for the ejector positioning means to movefrom one reservoir to another quickly and in a controlled manner,thereby allowing fast and controlled ejection of different fluids. Thatis, current commercially available technology allows the ejector to bemoved from one reservoir to another, with repeatable and controlledacoustic coupling at each reservoir, in less than about 0.1 second forhigh performance positioning means and in less than about 1 second forordinary positioning means. A custom designed system will allow theejector to be moved from one reservoir to another with repeatable andcontrolled acoustic coupling in less than about 0.001 second. In orderto provide a custom designed system, it is important to keep in mindthat there are two basic kinds of motion: pulse and continuous. Pulsemotion involves the discrete steps of moving an ejector into position,emitting acoustic energy, and moving the ejector to the next position;again, using a high performance positioning means with such a methodallows repeatable and controlled acoustic coupling at each reservoir inless than 0.1 second. A continuous motion design, on the other hand,moves the ejector and the reservoirs continuously, although not at thesame speed, and provides for ejection during movement. Since the pulsewidth is very short, this type of process enables over 10 Hz reservoirtransitions, and even over 1000 Hz reservoir transitions.

[0094] In order to ensure the accuracy of fluid ejection, it isimportant to determine the location and the orientation of the fluidsurface from which a droplet is to be ejected with respect to theejector. Otherwise, ejected droplets may be improperly sized or travelin an improper trajectory. Thus, another embodiment of the inventionrelates to a method for determining the height of a fluid surface in areservoir between ejection events. The method involves acousticallycoupling a fluid-containing reservoir to an acoustic radiation generatorand activating the generator to produce a detection acoustic wave thattravels to the fluid surface and is reflected thereby as a reflectedacoustic wave. Parameters of the reflected acoustic radiation are thenanalyzed in order to assess the spatial relationship between theacoustic radiation generator and the fluid surface. Such an analysiswill involve the determination of the distance between the acousticradiation generator and the fluid surface and/or the orientation of thefluid surface in relationship to the acoustic radiation generator.

[0095] More particularly, the acoustic radiation generator may beactivated so as to generate low energy acoustic radiation that isinsufficiently energetic to eject a droplet from the fluid surface. Thisis typically done by using an extremely short pulse (on the order oftens of nanoseconds) relative to that normally required for dropletejection (on the order of microseconds). By determining the time ittakes for the acoustic radiation to be reflected by the fluid surfaceback to the acoustic radiation generator and then correlating that timewith the speed of sound in the fluid, the distance—and thus the fluidheight—may be calculated. Of course, care must be taken in order toensure that acoustic radiation reflected by the interface between thereservoir base and the fluid is discounted. It will be appreciated bythose of ordinary skill in the art that such a method employsconventional or modified sonar techniques.

[0096] Once the analysis has been performed, an ejection acoustic wavehaving a focal point near the fluid surface is generated in order toeject at least one droplet of the fluid, wherein the optimum intensityand directionality of the ejection acoustic wave is determined using theaforementioned analysis optionally in combination with additional data.The “optimum” intensity and directionality are generally selected toproduce droplets of consistent size and velocity. For example, thedesired intensity and directionality of the ejection acoustic wave maybe determined by using not only the spatial relationship assessed asabove, but also geometric data associated with the reservoir, fluidproperty data associated with the fluid to be ejected, and/or by usinghistorical droplet ejection data associated with the ejection sequence.In addition, the data may show the need to reposition the ejector so asto reposition the acoustic radiation generator with respect to the fluidsurface, in order to ensure that the focal point of the ejectionacoustic wave is near the fluid surface, where desired. For example, ifanalysis reveals that the acoustic radiation generator is positionedsuch that the ejection acoustic wave cannot be focused near the fluidsurface, the acoustic radiation generator is repositioned usingvertical, horizontal and/or rotational movement to allow appropriatefocusing of the ejection acoustic wave.

[0097] In general, screening for the properties of the arrayconstituents will be performed in a manner appropriate to the type ofarray generated. Screening for biological properties such as ligandbinding or hybridization may be generally performed in the mannerdescribed in U.S. Pat. Nos. 5,744,305 and 5,445,934 to Fodor et al. U.S.Pat. Nos. 5,143,854 and 5,405,783 to Pirrung et al., and U.S. Pat. Nos.5,700,637 and 6,054,270 to Southern et al. Screening a substrate formaterial properties may be effected by measuring physical and chemicalproperties by routine methods easily adaptable to microarrays. Inaddition to bulk material characteristics or properties, surfacespecific properties may be measured by surface specific physicaltechniques and physical techniques that are adapted to surfacecharacterization. Macroscopic surface phenomena including adsorption,catalysis, surface reactions including oxidation, hardness, lubricationand friction, may be examined on a molecular scale using suchcharacterization techniques. Various physical surface characterizationtechniques include without limitation diffractive techniques,spectroscopic techniques, microscopic surface imaging techniques,surface ionization mass spectroscopic techniques, thermal desorptiontechniques and ellipsometry. It should be appreciated that theseclassifications are arbitrary made for purposes of explication, and someoverlap may exist.

[0098] It is to be understood that while the invention has beendescribed in conjunction with the preferred specific embodimentsthereof, the foregoing description is intended to illustrate and notlimit the scope of the invention. Other aspects, advantages andmodifications will be apparent to those skilled in the art to which theinvention pertains.

[0099] All patents, patent applications, journal articles and otherreferences cited herein are incorporated by reference in theirentireties.

[0100] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to implement the invention, and are not intended to limit the scopeof what the inventors regard as their invention. Efforts have been madeto ensure accuracy with respect to numbers (e.g., amounts, temperature,etc.) but some errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is in ° C.and pressure is at or near atmospheric.

EXAMPLE 1

[0101] This example describes preparation of an array ofoligonucleotides in the form of a library, and demonstrates the use offocused acoustic energy in the solid phase synthesis ofoligonucleotides.

[0102] Microporous glass, preferably controlled pore size glass (CPG),is sintered onto the surface of a glass plate to form a CPG layer havinga thickness sufficient to enable permeation to both the downward flowand the lateral wicking of fluids. Generally, a sufficient thickness isgreater than about 10 μm.

[0103] Accordingly, the CPG is applied to the glass surface at athickness of about 20 μm and the glass with powdered CPG residentthereon is heated at 750° C. for about 20 minutes then cooled.Commercially available microscope slides (BDH Super Premium 76×26×1 mm)are used as supports. Depending on the specific glass substrate and CPGmaterial used the sintering temperature and time may be adjusted toobtain a permeable and porous layer that is adequately attached to theglass beneath while substantially maintaining the permeability to fluidsand thickness of the microporous glass layer. The slides heated for 20minutes with a 1 cm square patch of microporous glass applied at apre-heating thickness of about 20 μm yield a sintered layer ofsubstantially the same depth as pre-heating, namely 20 μm.

[0104] The microporous glass layer is derivatized with a long aliphaticlinker that can withstand conditions required to deprotect the aromaticheterocyclic bases, i.e. 30% NH₃ at 55° C. for 10 hours. The linker,which bears a hydroxyl moiety, the starting point for the sequentialformation of the oligonucleotide from nucleotide precursors, issynthesized in two steps. First, the sintered microporous glass layer istreated with a 25% solution of 3-glycidoxypropyltriethoxysilane inxylene containing several drops of Hunig's base as a catalyst in astaining jar fitted with a drying tube, for 20 hours at 90° C. Theslides are then washed with MeOH, Et₂O and air dried. Neat hexaethyleneglycol and a trace amount of concentrated H₂SO₄ acid are then added andthe mixture is kept at 80° C. for 20 hours. The slides are washed withMeOH, Et₂O, air dried and stored desiccated at −20° C. until use.(Preparative technique generally described in British Patent Application8822228.6 filed Sep. 21, 1988.)

[0105] Focused acoustic ejection of about 0.24 pL of anhydrousacetonitrile (the primary coupling solvent) containing a fluorescentmarker onto the microporous substrate is then shown to obtain a circularpatch of about 5.6 μm diameter on the permeable sintered microporousglass substrate. The amount of acoustic energy applied at the fluidsurface may be adjusted to ensure an appropriate diameter of chemicalsynthesis for the desired site density. 5.6 μm diameter circular patchesare suitable for preparing an array having a site density of 10⁶sites/cm² with the circular synthetic patches spaced 10 μm apart centerto center, and the synthetic patches therefore spaced edge to edge atleast 4 μm apart at the region of closest proximity. All subsequentspatially directed acoustically ejected volumes in this example are ofabout 0.24 pL; it will be readily appreciated that the ejection volumescan be adjusted for solutions other than pure acetonitrile by adjustingthe acoustic energy as necessary for delivery of an appropriately sizeddroplet after spreading on the substrate (here about a 5 μm radius).

[0106] The oligonucleotide synthesis cycle is performed using a couplingsolution prepared by mixing equal volumes of 0.5 M tetrazole inanhydrous acetonitrile with a 0.2 M solution of the requiredβ-cyanoethylphosphoramidite, e.g. A-β-cyanoethylphosphoramidite,C-β-cyanoethylphosphoramidite, G-β-cyanoethylphosphoramidite, T(orU)-β-cyanoethylphosphoramidite. Coupling time is three minutes.Oxidation with a 0.1 M solution of I₂ in THF/pyridine/H₂O yields astable phosphotriester bond. Detritylation of the 5′ end with 3%trichloroacetic acid (TCA) in dichloromethane allows further extensionof the oligonucleotide chain. No capping step is required because theexcess of phosphoramidites used over reactive sites on the substrate islarge enough to drive coupling to completion. After coupling the slidethe subsequent chemical reactions (oxidation with I₂, and detritylationby TCA) are performed by dipping the slide into staining jars.Alternatively the focused acoustic delivery of I₂ in THF/pyridine/H₂Oand/or 3% TCA in dichloromethane to effect the oxidation and tritylationsteps only at selected sites may be performed if sufficient timetranspires to permit evaporation of substantially all the solvent fromthe previous step so that the synthetic patch edges do not move outwardsand closer to the neighboring synthetic patches, and further to providean anhydrous environment for subsequent coupling steps if I₂ inTHF/pyridine/H₂O is delivered within the reaction chamber.

[0107] After the synthesis is complete, the oligonucleotide isdeprotected in 30% NH₃ for 10 hours at 55° C. Because the couplingreagents are moisture-sensitive, and the coupling step must be performedunder anhydrous conditions in a sealed chamber or container. This may beaccomplished by performing the acoustic spotting in a chamber ofdesiccated gas obtained by evacuating a chamber that contains theacoustic ejection device and synthetic substrate and replacing theevacuated atmospheric gas with desiccated N₂ by routine methods; washingsteps may be performed in the chamber or by removing the slide andwashing it in an appropriate environment, for example, by a staining jarfitted with a drying tube. Because washing and other steps such asdetritylation may be more conveniently carried out outside the chamber,the synthesis may also be performed in a controlled humidity room thatcontains the controlled atmosphere chamber in which the spotting isdone, with the other steps carried out in the room outside the chamber.Alternatively, a controlled humidity room may be used for spotting withother steps carried out in less controlled environment by use of, forexample, a staining jar fitted with a drying tube.

EXAMPLE 2

[0108] This example describes preparation of a peptide array in the formof a combinatorial library, and demonstrates the use of focused acousticenergy in the combinatorial solid phase synthesis of all tetramers thatcan be made from the 20 naturally occurring amino acids (20⁴ or=160,000amino acid sequences in all) in a quadruplicate array format. Fouridentical copies of the combinatorial array to be prepared are containedin a 1 cm×1 cm area nominally divided into four quadrants, each quadrantcontaining 250,000 synthesized sites of size 10 μm×10 μm arrayed in 500rows and 500 columns. Only 400 rows and columns are used in eachquadrant; the first and last 50 rows and columns are not used forsynthesis, and function to space the four identical arrays from eachother and the edges of the area, although alternative arrangement of thefour identical arrays can obtain greater distance between arrays bymoving each array closer to the corners of the square area. In additionto systematically generating the combinatorial sequences, deposition ofthe monomers employs a systematic method of ensuring that similar aminoacid sequences are less likely to be spatially close. Although many suchmethods exist, with some requiring sophisticated computation, and cantake into account side chain similarities in addition to identity, e.g.hydrophobic Val, Leu, Ile the scheme used relies on a basic sequentiallist of amino acids which is phase shifted as the row number increases.For example the 20 natural amino acids can be listed sequentially basedon the alphabetic order of their single letter abbreviations, in whichcase: Ala (A) is “1”; Cys (C) is “2”; Asp (D) is 3; . . . Val (V) is“19”; and Trp (W) is “20”.

[0109] For the first monomer deposited, in the first row in a givenquadrant in which a peptide is synthesized, which is the 51^(st) nominalrow in that quadrant, beginning with the first synthetic column (51^(st)nominal column) amino acids (as activated for the synthesis described inmore detail below) are deposited as the basic sequential list from 1 to20 in alphabetical order of the one letter abbreviations. Beginning withthe second synthetic row (52^(nd) nominal row), the order is shifted byone position starting at “2” and returning to “1” after “20” (2, 3, 4, 5. . . 19, 20, 1); thus for the quadruplicate spaced array arrangementbeing made, in the 52^(nd) nominal row (second synthetic row) of a givenquadrant, the first amino acid deposited in the 51^(st) and 431^(st)nominal column of the 52^(nd) nominal row is “2” or Cys, and the aminoacids deposited in the 68^(th) and 448^(th), 69^(th) and 449^(th), and70^(th) and 450^(th) nominal columns of this row are 19, 20 and 1respectively (V, W, A).

[0110] Additional monomers are added in the quadrants as follows,although numerous alternatives exist. For the second monomer in thefirst synthetic row (51^(st) nominal row) the monomer deposition orderfor the second monomer is the same as for the first monomer in the first20 synthetic columns (nominal 51-70) of this row, and the order isshifted by one for each successive group of 20 synthetic columns, thusthe order is 2, 3 . . . 19, 20, 1 for nominal columns 71-90 (hereinafterdenoted [71-90]-{2, 3 . . . 19, 20, 1}) and according to this notation:[91-110]-{3, 4 . . . 20, 1,2}; [111-130]-{4,5 . . . 1,2,3} . . .[431-450]-{20, 1 . . . 17, 18, 19}. For the second and third monomers inthe second synthetic row (52^(nd) nominal row) the monomer depositionorder is shifted by one relative to the order for the underlying monomerin the first 20 synthetic columns (nominal 51-70) of this row, and theorder is shifted by one for each successive group of 20 syntheticcolumns, thus for the second monomer the order is 3, 4 . . . 20, 1, 2for nominal columns 51-70 and: [71-90]-{4, 5 . . . 1, 2, 3} [91-110]-{5,6 . . . 2, 3, 4}; [111-130]-6, 7 . . . 3, 4, 5}. . . [431-450]-{2, 3 . .. 19, 20, 1}. Note that for the second monomer of the second syntheticrow, the shift relative to the order of the first monomer in the firstmonomer in the first 20 columns of the first row ({1,2 . . . 18, 19,20}), is 2 because one is the shift between subsequent monomers (1^(st)

2^(nd); 2^(nd)

3^(rd)) and the first monomer of the second synthetic row is shifted byone relative to the first monomer of the first synthetic row. For thesecond and third monomers in the third synthetic row (53^(rd) nominalrow) the monomer deposition order is shifted by two relative to theorder for the underlying monomer in the first 20 synthetic columns(nominal 51-70) of this row, and the order is shifted by one for eachsuccessive group of 20 synthetic columns, thus the order for the secondmonomer is 5 . . . 20, 1, 2, 3, 4 for nominal columns 51-70 and:[71-90]-{6 . . . 1, 2, 3, 4, 5}, [91-110]{7, . . . 2,3,4, 5, 6},[111-130]-{8, . . . 4,5, 6,6, 7} . . . [431-450]-{4, . . . 19,20, 1,2,3}. For the second monomer in the Nth synthetic row (nominal row=50+N)the monomer deposition order for the second monomer is shifted by (N−1)relative to the order for the first monomer in the first 20 syntheticcolumns (nominal 51-70) of this row, and the order is shifted by one foreach successive group of 20 synthetic columns, thus (for (k*N+a) >20,(k*N+a) is shifted as beginning with N+a−20*I, where I is the integerdividend of the quotient of (k*N+a) and 20, representing number ofcycles with each integral multiple of 20 representing unshifted) theorder for the second monomer is (2*N−1), 2*N . . . (2*N−3), (2*N−2) fornominal columns 51-70 and: [71-90]-{(2*N . . . (2*N−2), (2*N−1)},[91-110]-{(2*N+1), (2*N+2) . . . (2*N−1), 2*N}, [111-130]-{(2*N+2),(2*N+3) . . . 2*N, (2*N+1)}. . . [431-450]-{(2*N−2), (2*N−1) . . .(2*N−4), (2*N−3)}. Thus for the second monomer in the 400^(th) syntheticrow (450^(th) nominal row) the monomer deposition order for the secondmonomer begins with 19 (799-780) is circularly shifted by 18 relative tothe order for the first monomer in the first 20 synthetic columns(nominal 51-70) of the first row, and the order is shifted by one foreach successive group of 20 synthetic columns, thus the order is 19, 20. . . (17), (18) for nominal columns 51-70 and: [71-90]-{20, 1 . . . 17,18, 19}, [91-110]-{1, 2 . . . 18, 19, 20}, [111-130]-{2, 3 . . . 19, 20,1} . . . [431-450]-{20, 1 . . . 17, 18, 19}. Note that for the secondmonomer of the Nth synthetic row, the shift relative to the order of thefirst monomer in the in the first 20 synthetic columns of the first row({1, 2 . . . 18, 19, 20}), is 2*(N−1) because (N−1) is the shift betweensubsequent monomers (1^(st)

2^(nd); 2 ^(nd)

3^(rd)) and the first monomer of a synthetic row N is shifted by (N−1)relative to the first monomer of the first synthetic row.

[0111] The synthetic chemical steps are modified from known solid phasesynthetic techniques (as described, for example, in Geysen et al.,International Patent Application PCT/AU84/00039, published as WO84/83564) that are adapted from the pioneering solid phase peptidesynthesis of Merrifield et al. ((1965) Nature 207:(996):522-23; (1965)Science 150(693)178-85; (1966) Anal. Chem. 38(13):1905-14; (1967)Recent. Prog. Horm. Res. 23:451-82). The conventional methods of solidphase peptide synthesis as taught in these seminal papers are describedin detail in Ericksen, B. W. and Merrifield, R. B. (1973) The Proteins2:255-57 Academic Press, New York, and Meinhofer, J. (1976) The Proteins2:45-267 Academic Press, New York. Briefly, all these methods add aminoacid monomers protected by tert-butoxycarbonyl (t-butoxycarbonyl, t-Boc)at their amino groups, including their alpha amino groups (N^(α)) to anascent peptide that is attached to the substrate at thecarboxy-terminal (C-terminal). The carbonyl moiety of the N^(α)-t-Bocamino acid to be added to the peptide is activated to convert thehydroxyl group of the carboxylic moiety into an effective leaving group,resembling an acid anhydride in reactivity, usingdicyclohexylcarbodiimide (DCC) to permit nucleophilic displacement bythe terminal N of the nascent peptide to form a peptide bond that addsthe monomer to the forming peptide. The newly added monomer has anN-terminus protected from further reaction by t-Boc, which is removedwith trifluoroacetic acid (TFA), rendering the terminal amino groupprotonated, followed by deprotonation of the terminal amino group withtriethylamine (TEA) to yield the reactive free amino group suitable foraddition of another monomer.

[0112] The substrate employed is polyethylene, although the classicsubstrate for solid phase peptide synthesis, divinylbenzene cross-linkedpolystyrene chloromethylated by Friedel-Crafts reaction of thepolystyrene resin on approximately one in four aromatic rings, couldalso be employed. Preparation of the polyethylene substrate, describedin Geysen et al., International Patent Application PCT/AU84/00039,published as WO 84/83564, involves γ-ray irradiation (1 mrad dose) ofpolyethylene immersed in aqueous acrylic acid (6% v/v) to yield reactivepolyethylene polyacrylic acid (PPA), according to the method ofMuller-Schulte et al. (1982) Polymer Bulletin 7:77-81.N^(α)-t-Boc-Lysine methyl ester is then coupled to the PPA by the Lysineε-amino side chain. After deprotection of the N^(α) by removal of thet-Boc with TFA followed by TEA, DCC/N^(α)-t-Boc-Alanine is added tocouple t-Boc-Ala to the N^(α) of the Lys, thereby forming a peptide likeN^(α)-t-Boc-Ala-Lys-ε-N-PPA linker to which the DCC activatedN^(α)-t-Boc-amino acid monomers can be sequentially added to form thedesired polymers upon deprotection of the N^(α) group of theN^(α)-t-Boc-Ala.

[0113] For an array format, and to increase the effective surface areafor polymer formation and enhance adhesion of acoustically ejectedreagent droplets to the synthetic substrate, polyethylene fiber sheetmaterial, approximate thickness 25 μm, available commercially andprepared by conventional methods is heat or fusion bonded according toroutine methods to a smooth polyethylene backing approximately 0.15 cmthick to form a polyethylene fiber coated rough permeable substrate. Thefiber coated sheet s cut into strips having the approximate dimensionsof a commercial slide, and γ-irradiated (1 mrad) in 6% v/v aqueousacrylic acid to form the PPA activated substrate. The substrate must beadequately dried because the t-Boc protected and DCC activated reagentsare water sensitive, and water contamination of acids applied to thesynthetic sites, such as TFA application can hydrolyze the peptide bond.Thus anhydrous synthetic conditions are required throughout.Conventional drying of the substrate is effected with warm dry air atatmospheric or subatmospheric pressure by routine methods, specifically,the slides are washed with MeOH, Et₂O, air dried and stored desiccatedat −20° C. until use.

[0114] The sequential combinatorial addition of monomers is performed asdescribed above with all sites spotted with the appropriateDCC/N^(α)-t-Boc-amino acid. The appropriate volume for acoustic ejectionis as above. This yields a quasi-parallel synthesis because the spottingof different sites is not simultaneous, but the can be modified tosynthesize the desired peptides only at some sites and synthesize atother sites later. The actual synthesis requires anhydrous organicsolvent washing steps to remove unreacted activated amino acids or TFAor TEA, for a total of 11 steps per monomer addition. Thus a completelysequential synthesis would increase the number of steps performed forsynthesizing an array drastically, but, for example synthesizing only atevery other site in a first synthetic round and then synthesizing in asecond session would improve array quality and only double the number ofsteps. To ensure that peptides are only formed at the chosen sites, theN^(α)-t-Boc-Ala-Lys-ε-N-PPA linker can be selectively deprotected toexpose the N^(α) of Ala only at chosen sites, by selective acousticenergy directed ejection of TFA onto the desired sites, followed bywashing and selective application of TEA, followed by washing to effect,for example, selective deprotection of every other site.

[0115] The basic quasi-parallel combinatorial synthesis of alltetra-peptides that can be made from the naturally occurring amino acidsmay be performed in 44 steps excluding substrate preparation. As noselective linker deprotection is required, the substrate is immersed inTFA in a staining jar fitted with a drying tube, then washed, andimmersed in TEA, and washed again, all under anhydrous conditions. Thesynthesis must be carried so that ejection of the fluid droplets occursin a controlled atmosphere that is at minimum dry, and inert to thereagents used. This may be obtained by performing the acoustic spottingin a chamber of desiccated gas obtained by evacuating a chamber thatcontains the acoustic ejection device and synthetic substrate andreplacing the evacuated atmospheric gas with desiccated N₂ by routinemethods; washing steps may be performed in the chamber or by removingthe slide and washing it in an appropriate environment, for example, bya staining jar fitted with a drying tube. Because washing and othersteps such as detritylation may be more conveniently carried out outsidethe chamber, the synthesis may also be performed in a controlledhumidity room that contains the controlled atmosphere chamber in whichthe spotting is done, with the other steps carried out in the roomoutside the chamber. Alternatively, a controlled humidity room may beused for spotting with other steps carried out in less controlledenvironment by use of, for example, a staining jar fitted with a dryingtube.

[0116] Use of pre-synthesized short oligopeptides can also be used inlieu of amino acid monomers. Since focused acoustic ejection enables therapid transition from the ejection of one fluid to another, manyoligopeptides can be provided in small volumes on a single substrate(such as a microtiter plate) to enable faster assembly of amino acidchains. For example, all possible peptide dimers may be synthesized andstored in a well plate of over 400 wells. Construction of the tetramerscan than be accomplished by deposition of only two dimers per site and asingle linking step. Extending this further, a well plate with at least8000 wells can be used to construct peptides with trimers.

EXAMPLE 3

[0117] Combinatorial methods of the preceding Examples 1 and 2 can beadapted to form combinatorial arrays of polysaccharides according to theinstant invention. In oligosaccharides, the monosaccharide groups arenormally linked via oxy-ether linkages. Polysaccharide ether linkagesare difficult to construct chemically because linking methods arespecific for each sugar employed. The ether oxygen linking group is alsosusceptible to hydrolysis by non-enzymatic chemical hydrolysis. Thus,there are no known methods of automated syntheses for ether linkedcarbohydrates, and conventional methods of making combinatorial arraysare not sufficiently flexible to permit combinatorial arrays ofpolysaccharides. The flexibility of acoustic spotting can be adapted toform oxy-ether linkage based combinatorial arrays by analogy to thealternative method of selective deblocking that may be employed formaking the arrays of Examples 1 and 2. That is, the specific chemicalmethods for forming the linkage between any pair of sugars may beconveniently selected so that a different solution is ejected for addinga glucose to a specific terminal sugar of the forming polysaccharide,such as fructose, than is ejected for adding glucose to a differentterminal sugar, such as ribose, without increasing the number of stepsinvolved as would be the case with photolithographic synthesis, andmight be the case with parallel printing of multiple reagents throughconventional multi nozzle ink-jet type printers. The resultingpolysaccharides remain susceptible to hydrolysis.

[0118] Polysaccharides may be synthesized in solution rather than thesolid phase, as can the biomolecules made in the preceding examples, andthe acoustic ejection of droplets can effect the solution syntheses ofarrayed polysaccharides at high density on a substrate without anyattachment during polymer formation by selective application ofdeblocking reagents to different sites. In situ solid phase synthesis ismore readily adaptable to automation of even oxy-ether linkage basedpolysaccharides because at least the deblocking steps may be donesimultaneously for all sites, although the susceptibility of thedifferent linkages to hydrolysis may affect overall yield for differentmonomer sequences differently. Recently, methods of replacing theoxy-ether with a thio-ether linkage (U.S. Pat. Nos. 5,780,603 and5,965,719) and with an amide linkage with the N atom linked to theanomeric C of the sugar (U.S. Pat. No. 5,756,712) have been introduced.The solid phase synthetic methods of the thioether linkage methods maybe directly adapted to form high density combinatorial arrays in ananalogous manner as techniques for the Merrifield peptide synthesis.Similarly, the amide linkage based polysaccharides may be adapted forsolid phase high density array formation by employing, for example thethioether based substrate linkage taught in U.S. Pat. Nos. 5,780,603 and5,965,719, or an amide linkage to an appropriate moiety functionalizedsurface by analogy to the linkage of U.S. Pat. No. 5,756,712.

[0119] Only the thioether based substrate linkage will be exemplified indetail, and this linkage will be used to make thioether (amide basedoligosaccharides may be made analogously by reference to U.S. Pat. No.5,756,712 with a thioether, or other, substrate linkage) basedcombinatorial array of oligosaccharides. The classic substrate for solidphase peptide synthesis, divinylbenzene cross-linked polystyrenechloromethylated by Friedel-Crafts reaction of the polystyrene resin onapproximately one in four aromatic rings is employed, although apolyethylene substrate may be substituted.

[0120] Spun polystyrene sheet made by conventional methods or obtainedcommercially is heat or fusion bonded to a polystyrene backing to yielda porous permeable layer of spun polystyrene of approximately 25 μmthickness. The appropriate extent of crosslinking and chloromethylationis effected by conventional chemical synthetic methods as required. Thethickness of the permeable layer will be appreciated to affect thedimensions of the area of actual chemical synthesis, as more verticalwicking room will result in less lateral spread of the acousticallydeposited reagents. It also will be appreciated that the extent ofcrosslinking may be adjusted to control the degree of swelling, andsoftening upon application of organic solvents, and that the fibrousnature of the porous, permeable layer of spun polystyrene providesrelatively more synthetic surface per nominal surface area of thesubstrate than provided by beads, thus less swelling is required toexpand synthetic area to polymer sites inside the fibers. The substrateis aminated by conventional chemical synthetic methods, washed andstored desiccated at −20° C. until use.

[0121] The linking of a sugar to this substrate is first effected.Succinic anhydride (1.2 equivalents) is added to a solution of1,2:3,4-di-O-isopropylidene-D-galactopyranose (1 equivalent) in pyridineat room temperature. The reaction is stirred overnight then concentratedin vacuo to yield1,2:3,4-di-O-isopropylidene-6-O-(3-carboxy)propanoyl-D-galactopyranose.80% aqueous acetic acid is added to the residue to remove theisopropylidene groups. When this reaction is complete, the reactionmixture is concentrated in vacuo. Excess 1:1 acetic anhydride/pyridineis then added to the residue to form1,2,3,4-O-acetyl-6-O-(3-carboxy)propanoyl-D-galactopyranose, to whichexcess thiolacetic acid in dry dichloromethane under argon at 0° C. andBF₃ etherate is then added. The cold-bath is removed after 10 minutes.After 24 h the mixture is diluted with dichloromethane, washed withsaturated sodium bicarbonate, dried over sodium sulfate, andconcentrated to yield1-S-acetyl-2,3,4-tri-O-acetyl-6-O-(3-carboxy)propanoyl-1-thio-α-D-galactopyranose.The aminated polystyrene (Merrifield resin) substrate is contacted withthe1-S-acetyl-2,3,4-tri-O-acetyl-6-O-(3-carboxy)propanoyl-1-thio-α-D-galactopyranoseand a carbodiimide coupling reagent to afford the O,S-protectedgalactopyranose coupled to the substrate through the6-O-(3-carboxy)propanoyl group.

[0122] The preceding substrate is used for combinatorial synthesis ofthioether linked polysaccharides based on thiogalactose derivatives.Nine copies of the combinatorial array of all possible trimers of fourmonomeric 1-thiogalactose derivatives (4³=64 in all) are synthesized ona total substrate surface area of 1 cm² divided into square syntheticsites 333 μm×333 μm, corresponding to a site density of 1000 sites/cm².This arrangement permits a 3 site or 999^(μm) spacing between each copyof the array in each axis of the array plane. A 25 pL droplet offluorescent solvent deposited on the described porous permeable spunpolystyrene on polystyrene substrate yields a spot of about 56 μmdiameter, and a 100 pL droplet yields a spot of about 112 μm diameter(cylindrical shaped spot wicked into depth of porous substrate withabout ½ of porous layer occupied by solid polystyrene and littleswelling thereof).

[0123] Step A—Synthesis of1-Dithioethyl-2,3,4,6-tetra-O-acetyl-galactopyranoside:

[0124] 1-Thio-2,3,4,6-tetra-O-acetyl-galactopyranoside (500 mg, 1.37mmol) and diethyl-N-ethyl-sulfenylhydrazidodicarboxylate (360 mg, 2.0mmol) (prepared by known methods as described by Mukaiyama et al. (1968)Tetrahedron Letters 56:5907-8) are dissolved in dichloromethane (14 mL)and stirred at room temperature. After 10 min, the solution isconcentrated and column chromatography (SiO₂, hexane/ethylacetate 2:1)yields 1-dithioethyl-2,3,4,6-tetra-O-acetyl-galactopyranoside (580 mg,quant) as a white solid (R_(f) 0.27 in hexanes/ethyl acetate(2:1)).¹H-NMR (360 MHZ, CHCl₃): .δ 1.30 (dd, 3H, J=7.4 Hz, CH₃), 1.96,2.02, 2.03, 2.13 (4 s, 12H, 4CH₃CO), 2.79 (ddd, 2H, J=7.4 Hz, J=7.4 Hz,J=1.3 Hz, CH₂), 3.94 (ddd, 1H, J₄,5=1.0 Hz, J₅,6a=6.6 Hz, J₅,6b=7.6 Hz,5-H), 4.10 ddd, 2H, 61-H, 6b-H), 4.51 (d, 1H, J₁,2=10.0 Hz, 1-H), 5.05(dd, 1H, J₂,3=10.0 Hz, J₃,4=3.3 Hz, 3-H)), 5.38 (dd, 1H, J₁,2=10.0 Hz,J₃,3=10.0 Hz, 2-H), 5.40 (dd, 1H, J₃,4=3.3 Hz, J₄,5=1.0 Hz, 4-H); m/zcalculated for C₁₆H₂₄O₉S₂ (M+Na) 447.1, found 447.0.

[0125] Step B—Synthesis of 1-dithioethyl-β-D-galactopyranoside:

[0126] 1-Dithioethyl-2,3,4,6-tetra-O-acetyl-galactopyranoside from StepA (500 mg, 1.18 mmol) is dissolved in dry methanol (10 mL) and treatedwith methanolic sodium methoxide (1 M, 150 μL). After 2 h, the solutionis neutralized with Amberlite 1R-120 (H⁺) resin, filtered andconcentrated to give 1-dithioethyl-6-β-D-galactopyranoside as a whitesolid (300 mg, quant).

[0127] Step C—Coupling of l-Dithioethyl-β-D-galactopyranoside to theSubstrate:

[0128] 1-Dithioethyl-6-O-D-galactopyranoside (200 mg, 780 μmol) isdissolved in dry pyridine (8 mL), and DMAP (5 mg) is added to themixture, which is maintained at 60° C. throughout.

[0129] Of the total (9×64=576) sites used to form the 9 duplicatearrays, and in each duplicate array of 64 sites of actual synthesis,{fraction (1/4 )}(16 per array, 144 total) of the array sites arepatterned with the 1-dithioethyl-6-β-D-galactopyranoside/DMAP. in drypyridine. This solution is acoustically ejected onto the substrate atthe desired locations. Dry controlled atmospheric conditions, namely adry inert gas environment, are also used for this oligosaccharidesynthesis. The appropriate volume deposited at each site is determinedby test deposition at some of the array sites, taking into considerationthat the synthetic area should be wholly contained in the syntheticsite, and too much dead space is preferably avoided. About 10 to 100 pLdroplet volumes are found to be appropriate, and 100 pL is spotted ontothe sites where the first monomer is desired to be1-dithioethyl-6-β-D-galactopyranoside. The substrate is as described,spun polystyrene resin on a polystyrene backing (trityl chloride-resin,loading 0.95 mmol/g of active chlorine, polymer matrix: copolystyrene-1%DVB) is heated for 24 h at 60° C. The resin is filtered off, and washedsuccessively with methanol, tetrahydrofuran, dichloromethane and diethylether (10 mL each) to afford 1-dithioethyl-6-β-D-galactopyranosidecovalently linked to the trityl resin through the hydroxyl group in the6-position at the desired sites.

[0130] Step D—Patterning Additional 1-Dithioethyl-6-pyranosides:

[0131] It will be readily appreciated that this step can be practicedwith other 1-dithioethyl-6-pyranosides as desired to be linked to thesubstrate. ¼ of the sites of each of the duplicate arrays are spottedwith a solution for linking 1-dithioethyl-6-β-D-glucopyranoside in aboutthe same volume as deposited in Step C, ¼ are spotted to yield the1-dithioethyl-6-β-D-mannopyranoside, and the remaining ¼ are spotted toyield the 1-dithioethyl-6-β-D-allopyranoside.

[0132] Step E—Generation of the Free Thiol on the Substrate:

[0133] The substrate sites from Step C spotted with dry tetrahydrofuran(THF) in the area of 1-dithioethyl-6-pyranoside deposition (about 4 pLper pL deposited in Step C). Dry methanol (about ¾ pL per pL depositedin Step C), dithiothreitol (about 185 picograms) and triethylamine(about ½ pL per pL deposited in Step C) are deposited at desiredsynthetic areas of the combinatorial sites by acoustic deposition andthe sites are allowed to react under the specified controlled atmosphereconditions for about 10 minutes to an hour at room temperature. Theentire substrate is washed by immersion in an adequate volume,successively, of methanol, tetrahydrofuran, dichloromethane and diethylether. Micro-FTIR (of substrate deposition sites): 2565 cm⁻¹ (SHstretch). Alternatively, if selective generation of the free thiol isnot desired, the substrate may be treated on the whole of the surface asfollows: 8 ml dry THF is applied to the surface of the substrate whichis placed in a shallow container just large enough to contain thesubstrate, 1.2 ml dry ethanol, 256 mg dithiothreitol, and 0.8 mltriethylamine are added to the THF and the container is shaken for about10 hours at room temperature under the described conditions.

[0134] Step F—Michael Addition Reaction:

[0135] The substrate from Step E is again placed in the shallowcontainer of Step E and swollen in dry N,N-dimethylformamide (4 mL) andthen cyclohept-2-en-1-one (280 μl, 252 μmol) is added and the containeris shaken at room temperature. After 2 hours, the liquid is removed andthe substrate is washed successively with methanol, tetrahydrofuran,dichloromethane and diethyl ether (40 mL each). Alternatively ifselective Michael addition is desired, the desired sites may beselectively spotted in the area of synthesis: N,N-dimethylformamide(about 2.5 pL per pL deposited in Step C); cyclohept-2-en-1-one (about0.2 pL, 0.2 picomole per pL deposited in Step C). The selectivelyspotted sites are allowed to react under the specified controlledatmosphere conditions for about 10 minutes to an hour at roomtemperature prior to the specified washing steps.

[0136] Step G—Reductive Amination with an Amino Acid:

[0137] The substrate from Step F is again placed in the shallowcontainer of preceding steps and swollen in dichloromethane (4 mL).Glycine tert-butyl ester hydrochloride (150 mg, 1,788 μmol), sodiumsulfate (400 mg), sodium triacetoxyborohydride (252 mg, 1188 μmol) andacetic acid (40 μL) are added at room temperature under argon atmosphereand the container shaken for 24 hours. The liquid is removed and thesubstrate is washed successively with washed successively with water,methanol, tetrahydrofuran and dichloromethane.

[0138] Additional monomers may be added by repetition of the precedingsteps with the desired 1-dithioethyl-6-pyranosides. It will be readilyappreciated that this step can be practiced with1-dithioethyl-6-β-D-galactopyranoside/DMAP and the other1-dithioethyl-6-pyranoside/DMAP desired for linking to the substrate.The desired sites of each of the duplicate arrays are selectivelyspotted with the appropriate 1-dithioethyl-6-pyranoside/DMAP solutionfor linking in about the same volume as deposited in Step C(1-dithioethyl-6-β-D-mannopyranoside/DMAP,1-dithioethyl-6-β-D-allopyranoside/DMAP, and1-dithioethyl-6-β-D-glucopyranoside/DMAP).

EXAMPLE 4

[0139] Combinatorial arrays of alloys can readily be prepared using themethodology of the invention. Molten metals are acoustically ejectedonto array sites on a substrate. No monomer sequence exists for metals,but the composition of the alloys may be altered by deposition of moreof a given metal at a certain site without problems associated withpolymer elongation; the problem with deposition of more metal dropletsof the same volume to form different compositions is that array densitymust be decreased to accommodate the most voluminous composition made,as the size of droplets is not conveniently adjusted over wide ranges ofdroplet volume. An additional reason to reduce array density in alloyformation is that with alloys it is often desirable to form a materialthat has a bulk and surface, rather than a film which has a surface butnot a bulk and therefore the properties of the thin-layer “surface” arenot the same as the surface of the bulk material (see generallySomorjai, Surface Chemistry and Catalysis, supra).

[0140] As may be readily appreciated, an infinite number of compositionsof any two metals exist. Composition in terms of combinatorial synthesisof arrays of alloys by acoustic ejection of fluid is complicated by thevolumetric acoustic ejection being different for different molten metalshaving different densities and interatomic interactions, but thedifferent stoichiometric compositions generated correspond to differentcombinations of metal and number of droplets deposited are reproducible,e.g. an alloy of 5 droplets of Sn ejected at an energy, E₁ and fivedroplets of Cu ejected at E₁ or E₂ will have the same compositions whenduplicated under the same conditions, and the stoichiometric compositionof alloys of interest can always be determined by SIMS. To promoteuniform alloy formation it is desirable to spot all the droplets ofmolten metal to be deposited onto a site in rapid succession rather thanwaiting for a droplet to solidify before depositing another, althoughsuch combinatorial “stacks” are also of potential interest. As it ismost convenient not to change acoustic energy between deposition ofdroplets, the same energy is most conveniently used for ejectingdifferent metals, and the stoichiometric and other, including surfaceproperties of the material so generated may be determined later andreproduced by exact duplication of the synthetic process. The moltenmetals must be at an appropriate temperature (T) above its melting pointto ensure that the droplet is still molten when it reaches thesubstrate. In addition to an inert gas environment, which may beappreciated to be important if making alloys rather than stacks ofoxidized metal salts is desired, to prevent oxidation of the metalsespecially at the surface of the droplets, a gas with low heat capacityis preferable to high heat capacity gases. In addition, the temperatureof the substrate and the distance between the substrate and the fluidmeniscus may be adjusted to ensure molten material reaching thesubstrate and remaining molten for sufficient time to permit alloyingwith subsequently deposited droplets. Furthermore, after a given alloycomposition is made at a given array site, both the ejection energy andthe meniscus to substrate distance may require adjustment in light ofthe foregoing considerations, as is readily appreciated.

[0141] A convenient systematic combinatorial approach involves selectinga number of molten compositions for ejection and a total number ofdroplets deposited at each site. Array density of 10⁵ sites/cm² isconvenient as each site is conveniently a 100 μm square, an area whichcan be easily appreciated to accommodate 10, approximately picoliter(pL) sized, droplets, because 10 pL spread uniformly over the area ofthe site would be only 1 μm, deep, and gravity prevents such completespreading and low surface angle.

[0142] For 4 different molten metallic compositions available forejection and 10 droplets, it may easily be demonstrated that 342possible compositions exist, and likewise for 15 droplets, 820 possiblecompositions exist in terms of droplet number. For d dropletcompositions with m ejected metals (although the molten ejection vesselcontents need not be a pure metal, and may themselves be an alloy):

^(d) Q _(m)=_(n=1→m)Σ(S(m)_(n))*(Z(m,d)_(n))

[0143]^(d)Q_(m) is defined as # metal compositions for d=# droplets, m=#of molten compositions available to be ejected; S(m)_(n) is the # ofunique sets having n members of the m available molten compositions;Z(n,d)_(n) is # of d droplet combinations of n used of the m availablefor deposition, corresponding to S(m)_(n). Further:

Z(m,d)_(n)=_(i=1→C(nd)) ΣO(n,d)_(i)

[0144] CS(n,d)_(,i) denotes ith set of coefficients for n componentsthat add to d droplets, with C(n,d), representing the total number ofcoefficient sets satisfying this requirement; O(n,d)_(i) is the numberof possible orderings of the ith set of n coefficients for d dropletscorresponding to CS(n,d)_(,i).

[0145] For example, for d=10, m=4, let the 4 vessels contain,respectively, Sn, In, Cd and Zn.

[0146] 1 metal compositions (n=1):

[0147] Z(4,10)₁=_(i=1→C(1,10))ΣO(1,10)_(i)=1*1, because the onlypossible coefficient is 10, and it can be ordered in only one way. Thecorresponding S(4)₁ is 4, as 4 unique sets of 1 metal can be chosen forejection.

[0148] 2 metal compositions (n=2):

[0149] The corresponding S(4)₂ is 6, as [4!/2!]/2! unique sets of 2metals can be chosen for ejection. The C(2,10) unique sets of 2non-negative, nonzero coefficients that add to 10, such as (9,1) and thecorresponding O (2,10)_(i) are [denoted by the notation{CS(2,10)₁:O(2,10)₁, CS(2,10)₂, 1:O(2,10)₂ . . .CS(2,10)_(C(n,d)):O(2,10)_(C(n,d))}]:

[0150] {(9,1):2, (8,2):2, (7,3):2, (6,4):2, (5,5):1};

Z(4,10)₂=_(i=1→C(2,10))ΣO(2,10)_(i)=2+2+2+2+1=9.

[0151] 3 metal compositions:

[0152] The corresponding S(4)₃ is 4 ([4!/1!]/3!), 4 unique sets of 3metals can be chosen for ejection. The C(3,10) unique sets of 3non-negative, nonzero coefficients that add to 10 are: {(8,1,1):3,(7,2,1):6, (6,3,1):6, (6,2,2):3, (5,4,1):6, (5,3,2):6, (4,4,2):3,(4,3,3):3};

[0153] Z(4,10)₃=_(i=1→C(3,10))ΣO(3,10)i=3+6+6+3+6+6+3+3=36.

[0154] 4 metal compositions:

[0155] The corresponding S(4)₄ is 1 (4!/4!), as 1 unique sets of 4metals can be chosen for ejection. The C(4,10) unique sets of 4non-negative, nonzero coefficients that add to 10 are: {(7,1,1,1):4,(6,2,1,1):12, (5,3,1,1):12, (5,2,2,1):12, (4,4,1,1):6, (4,3,2,1):24,(4,4,2,2):6, (3,3,3,1):4, (3,3,2,2):6};

[0156] Z(4,10)₄=_(i=1→C(4,10))ΣO(4,10)i=4+12+12+12+6+24+6+4+6=86.

[0157] From the preceding:

[0158]¹⁰Q₄=_(n=1→4)Σ(S(4)_(n))*(Z(4,10)_(n))=4*1+6*9+4*36+1*86=288.

[0159] An appropriate substrate for the alloy array of acousticallydeposited molten metallic compositions is made of sintered alumina byconventional methods or obtained commercially. An array of Sn (mp=281.8°C.), In (mp=156.6° C.), Cd (mp=320.9° C.) and Zn (mp=419.6° C.)components (e.g. pure ejected molten metal compositions) is formed byacoustic deposition of 15 droplets/array site on a sintered aluminasubstrate. Thickness of the substrate is about 0.25 cm, to withstand theheat. The site density is chosen to allow all possible dropletcompositions that can be made from four metals with 15 droplets, 820possible compositions including, for example (in droplets): 14(Sn),1*(In); 12Sn, 1In, 1Cd, 1Zn; 1Sn, 12In, 1Cd, 1Zn. These compositions andthe 901 remaining compositions may be obtained as above demonstrated for10 droplet compositions of four components. The chosen density is 1000sites/cm2, corresponding to a nominal site size of 333×333 μm, andpermitting the complete collection of compositions to be made on a 1 cm²area. Duplicate copies of the array are made on a commercial microscopeslide sized strip of substrate, separated by ½ cm to permit theconvenient separation of the two identical arrays.

[0160] The acoustic energy is adjusted to yield an average dropletvolume of about 1 pL, and 15 droplet ejection that does not exceed the333×333 μm square area provided for the site, under the desiredconditions, including atmosphere pressure and composition, length ofdroplet flight, substrate temperature. After the average droplet size isadjusted to about one pL, 15 droplets of each metal are acousticallyejected onto a site and the ejection energy is adjusted downwards if anyof these pure sites exceed the margins of the site. Enough sites existfor all 820 possible compositions to be ejected onto each 1 cm squarearray after using up to 96 of the available 1000, sites for calibration,but the single ejected component sites so created may function as thesingle composition sites if sufficiently the localized region withinwhich the alloy resides similar to the other sites in dimension, asdimensions affect cooling and a substantially different geometry wouldnot be precisely the same material.

[0161] Although the actual volumes ejected of the different moltencomponents may be adjusted to be equal by using a different acousticenergy of ejection, more rapid ejection is possible if the ejectionenergy is held constant. It is readily apprehended that if too wide adiscrepancy exists between the droplet volumes ejected for eachcomponent, that the overall geometry of the cooling composition couldvary widely depending on its makeup, but this is not the case for themetals being deposited here, because both their densities and factorsdetermining interatomic interactions in the molten state, such aspolarizability, are sufficiently similar. In all cases the conditionsfor the formation of the alloy at a given site are always reproducible,and the actual composition and other physical properties of thecomposition may be ascertained by physical methods including alldescribed surface physical characterization methods.

[0162] Because of the toxicity of Cd, the acoustic deposition of themolten metals is carried out in a separate atmospherically controlledlow humidity chamber under Ar gas to reduce undesired reactions andcooling. Higher heat capacity inert gases and more reactive gases, suchas O₂, and O₂/hydrocarbons may be used for experiments under differentconditions, but may require adjustment of the distance between the fluidmeniscus and substrate or the temperature of the molten reagent to beejected or both to ensure that the droplet reaches the substrate in amolten state.

[0163] After calibration the first duplicate array is spotted byacoustic ejection as described onto a substrate maintained at atemperature of 125° C. Each of the 820 possible 15 droplet compositionsis made by sequentially depositing fifteen droplets at each site, the 15droplets deposited according to the different coefficient arrangementsdescribed above. The metals are maintained at a known temperature thatis sufficiently greater than the mp of the metal that the ejecteddroplet arrives at the substrate surface molten under the conditions,including distance of flight and pressure, temperature and heat capacityof the atmosphere. The droplets are deposited at each site lowestmelting metal first in order of increasing melting temperature with thehighest melting temperature metal deposited last, e.g., In, Sn, Cd, Zn,so that successive droplets of higher melting temperature metal willmelt any solidified material. The procedure is repeated at differentsubstrate temperatures at 5 degree intervals until arrays formed withsubstrate temperature ranging from 40° C. to 425° C. are formed.

We claim:
 1. A method for generating an array of molecular moieties on aporous substrate surface divided into a plurality of discrete surfacesites, the method comprising applying focused acoustic energy to each ofa plurality of a reservoirs each containing a molecular moiety in afluid, wherein the focused acoustic energy is applied using an acousticejector comprised of an acoustic radiation generator and a focusingmeans in a manner effective to eject a droplet from each reservoirtoward the substrate surface such that the molecular moiety in eachdroplet attaches to a localized region within a different surface site.2. The method of claim 1, wherein each molecular moiety is different. 3.The method of claim 2, wherein a droplet is ejected toward each surfacesite, such that every surface site has a molecular moiety attachedthereto.
 4. The method of claim 3, wherein each molecular moiety isdifferent.
 5. The method of claim 1, wherein the molecular moieties arebiomolecules.
 6. The method of claim 5, wherein the biomolecules arenucleotidic.
 7. The method of claim 6, wherein the biomolecules areoligonucleotides.
 8. The method of claim 7, wherein the biomolecules arenucleotidic monomers, and the method further comprises stepwisesynthesis of an oligonucleotide within each surface site by repeateddeposition of individual nucleotidic monomers at each site using focusedacoustic energy.
 9. The method of claim 5, wherein the biomolecules arepeptidic.
 10. The method of claim 3, wherein the porous substratesurface is comprised of at least 62,500 discrete surface sites.
 11. Themethod of claim 10, wherein the porous substrate surface is comprised ofat least 250,000 discrete surface sites.
 12. The method of claim 11,wherein the porous substrate surface is comprised of at least 1,000,000discrete surface sites.
 13. The method of claim 12, wherein the poroussubstrate surface is comprised of at least 1,500,000 discrete surfacesites.
 14. A molecular array comprised of a plurality of differentmolecular moieties on a porous substrate surface divided into aplurality of discrete surface sites, each site containing one molecularmoiety attached to the substrate surface in a localized region withinthe site, wherein the different sites are present at a density of atleast 62,500 sites per square centimeter.
 15. The molecular array ofclaim 14, wherein the density is at least 250,000 sites per squarecentimeter.
 16. The molecular array of claim 15, wherein the density isat least 1,000,000 sites per square centimeter.
 17. The molecular arrayof claim 16, wherein the density is at least 1,500,000 sites per squarecentimeter.
 18. The molecular array of claim 14, wherein each molecularmoiety is different.
 19. The molecular array of claim 13, wherein themolecular moieties are biomolecules.
 20. The molecular array of claim19, wherein the biomolecules are oligonucleotides.
 21. The moleculararray of claim 20, wherein the biomolecules are peptidic.